Kank proteins: a new family of ankyrin-repeat domain-containing proteins

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.     

microRNA-211 promotes proliferation,migration, and invasion ability of oral squamous cell carcinoma cells via targeting the bridging integrator 1 protein

Oral squamous cell carcinoma (OSCC), the most common pathological type of oral cancer, is still a frequent malignancy with unsatisfactory prognosis. Accumulating studies have proven some microRNAs (miRNAs) can function as oncogenes in OSCC by targeting tumor suppressors. In this study, we first investigated the expression and role of tumor suppressor bridging integrator-1 (BIN1) in OSCC tissues and cells. Our results indicated that BIN1 was low expressed in the OSCC tissues and cell lines (SCC6, SCC9, SCC25, HN4, and HN6) along with miR-211 was highly expressed in OSCC tissues and cell lines, and BIN1 overexpression could evidently inhibit their proliferation, migration, and invasion abilities. Next, we used bioinformation algorithms to predict the potential miRNA targeting BIN1 and chose miR-211 for further study. miR-211, a highly expressed miRNA in OSCC cells, could specifically bind with the 3′-untranslated region (3′-UTR) of BIN1 to trigger its degradation. Addition of miR-211 inhibitor could evidently suppress the malignant behaviors of OSCC cells by upregulating BIN1 expression and inhibit the activation of the EGFR/MAPK pathway. Taken together the findings of the study indicated that miR-211 mediated BIN1 downregulation had crucial significances in OSCC, suggesting the miR-211 might be a novel potential therapeutic target for the OSCC treatment.     

MTFP1 overexpression promotes the growth of oral squamous cell carcinoma by inducing ROS production

Mitochondrial fission process 1 (MTFP1) is a novel nuclear‐encoded protein that promotes mitochondrial fission. Increasing lines of evidence indicate that increased mitochondrial fission is involved in carcinogenesis and tumor progression. However, the expression and biological effects of MTFP1 in cancer development is still unclear, especially in oral squamous cell carcinoma (OSCC). In this study, we first evaluated the expression of MTFP1 in 12‐paired OSCC tumor and peritumor tissues. We then explored the effects of MTFP1 knockdown or overexpression on cell growth by cell proliferation, colony formation, cell cycle, and cell apoptosis assays. Furthermore, the mechanisms by which MTFP1 promoted OSCC cell growth were explored. Our results showed that MTFP1 is frequently overexpressed in OSCC tissues. Functional experiments revealed that MTFP1 promoted the growth of OSCC cells by inducing the progression of cell cycle and suppressing cell apoptosis. Mechanistically, MTFP1 overexpression‐mediated mitochondrial fragmentation and subsequent ROS production was found to be involved in the promotion of OSCC cell growth. Collectively, our study demonstrates that MTFP1 plays a critical oncogenic role in OSCC carcinogenesis, which may serve as a potential therapeutic target in the treatment of this malignance.     

TCRP1 promotes radioresistance of oral squamous cell carcinoma cells via Akt signal pathway

Tongue cancer resistance-associated protein 1 (TCRP1) is a novel gene located on human chromosome 11q13.4 which has been reported as a candidate related to chemotherapeutic resistance to cisplatin. Results suggest that TCRP also contribute to radioresistance in oral squamous cell carcinoma (OSCC) cells. We previously established exogenous overexpression of TCRP1 cell line Tca8113/TCRP1 and TCRP1 knockdown cell line Tca8113/PYM-siRNA and paired control cell lines, which provides a cell model system to investigate the roles and mechanisms of TCRP1-mediated radioresponse in OSCC. In this study, we first compared the radiosensitivity of up/down-regulating expression of TCRP1 cell lines and paired control cell lines by a clonogenic survival assay, Hoechst 33258 staining, cell growth assay, and comet assay. The results indicated that TCRP1 played a significant role in mediating OSCC radioresistance through decreased cells apoptosis and increased cellular proliferation and long-term survival. The further study found that TCRP1 function by up-regulating Akt activity and levels and then elevating the level of NF-κB. In summary, these results provided strong evidence for the linkage between TCRP1 and radiation sensitivity and may provide theoretical base of TCRP1 as a potential molecular mark of estimating the response for irradiation in OSCC.     

Ubiquitin D Promotes Progression of Oral Squamous Cell Carcinoma via NF-Kappa B Signaling

Ubiquitin D (UBD) is highly upregulated in many cancers, and plays a pivotal role in the pathophysiological processes of cancers. However, its roles and underlying mechanisms in oral squamous cell carcinoma (OSCC) are still unclear. In the present study, we investigated the role of UBD in patients with OSCC. Quantitative real-time polymerase chain reaction and Western blot were used to measure the expression of UBD in OSCC tissues. Immunohistochemistry assay was used to detect the differential expressions of UBD in 244 OSCC patients and 32 cases of normal oral mucosae. In addition, CCK-8, colony formation, wound healing and Transwell assays were performed to evaluate the effect of UBD on the cell proliferation, migration, and invasion in OSCC. Furthermore, a xenograft tumor model was established to verify the role of UBD on tumor formation in vivo. We found that UBD was upregulated in human OSCC tissues and cell lines and was associated with clinical and pathological features of patients. Moreover, the overexpression of UBD promoted the proliferation, migration and invasion of OSCC cells; however, the knockdown of UBD exerted the opposite effects. In this study, our results also suggested that UBD promoted OSCC progression through NF-κB signaling. Our findings indicated that UBD played a critical role in OSCC and may serve as a prognostic biomarker and potential therapeutic target for OSCC treatment.     

Angiotensin II directly impairs adipogenic differentiation of human preadipose cells

microRNAs (miRNAs) are small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. Reduced expression of miR-338 has been reported in several types of cancers; however, the role of miR-338 in oral squamous cell carcinoma (OSCC) has not been elucidated. In this study, we demonstrated that miR-338 was dramatically downregulated in OSCC tissues and cell lines. Overexpression of miR-338 significantly inhibited proliferation, colony formation, migration, and invasion of OSCC cells. In addition, neuropilin1 (NRP1) was identified as a target of miR-338 in OSCC cells and inversely correlated with miR-338 in OSCC tissues. Furthermore, restoration of NRP1 attenuated the tumor-suppressive effects of miR-338. Taken together, miR-338 might inhibit growth and metastasis of OSCC cells by targeting NRP1.     

PER1 suppresses glycolysis and cell proliferation in oral squamous cell carcinoma via the PER1/RACK1/PI3K signaling complex

There is increasing evidence that the core clock gene Period 1 (PER1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of PER1 in promoting tumor progression remain largely unknown. Here, we discovered that PER1 was markedly downregulated in oral squamous cell carcinoma (OSCC). Then, OSCC cell lines with stable overexpression, knockdown, and mutation of PER1 were established. We found that PER1 overexpression significantly inhibited glycolysis, glucose uptake, proliferation, and the PI3K/AKT pathway in OSCC cells. The opposite effects were observed in PER1-knockdown OSCC cells. After treatment of PER1-overexpressing OSCC cells with an AKT activator or treatment of PER1-knockdown OSCC cells with an AKT inhibitor, glycolysis, glucose uptake, and proliferation were markedly rescued. In addition, after treatment of PER1-knockdown OSCC cells with a glycolysis inhibitor, the increase in cell proliferation was significantly reversed. Further, coimmunoprecipitation (Co-IP) and cycloheximide (CHX) chase experiment demonstrated that PER1 can bind with RACK1 and PI3K to form the PER1/RACK1/PI3K complex in OSCC cells. In PER1-overexpressing OSCC cells, the abundance of the PER1/RACK1/PI3K complex was significantly increased, the half-life of PI3K was markedly decreased, and glycolysis, proliferation, and the PI3K/AKT pathway were significantly inhibited. However, these effects were markedly reversed in PER1-mutant OSCC cells. In vivo tumorigenicity assays confirmed that PER1 overexpression inhibited tumor growth while suppressing glycolysis, proliferation, and the PI3K/AKT pathway. Collectively, this study generated the novel findings that PER1 suppresses OSCC progression by inhibiting glycolysis-mediated cell proliferation via the formation of the PER1/RACK1/PI3K complex to regulate the stability of PI3K and the PI3K/AKT pathway-dependent manner and that PER1 could potentially be a valuable therapeutic target in OSCC.Subject terms: Oral cancer, Cell growth, RNAi     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

IEX-1相互作用蛋白的筛选与鉴定

骨肉瘤即原发于骨的恶性肿瘤,易发生早期肺转移且预后差,恶性程度高.本研究小组前期研究发现,IEX-1在骨肉瘤中具有重要作用.为了阐明其作用机制,本研究运用酵母双杂交技术筛选其相互作用蛋白,共鉴定出12个IEX-1相互作用蛋白,包括生物氧化相关酶类、分子伴侣、信号转导相关蛋白等.并首次证实clusterin(CLU,又名载脂蛋白J)与IEX-1存在相互作用,两者在细胞中具有很好的共定位.采用RNAi干扰减低骨肉瘤细胞中内源性CLU表达水平,显著抑制了细胞增殖与细胞侵袭能力.为阐明IEX-1在骨肉瘤发生发展中的作用机制提供了重要的线索,为骨肉瘤的早期诊治及预后监测提供了新的靶点.     

Downregulation of Microrna‐148a in Cancer‐Associated Fibroblasts from Oral Cancer Promotes Cancer Cell Migration and Invasion by Targeting Wnt10b

It is well established that crosstalk between cancer‐associated fibroblasts (CAFs) and cancer cells plays a critical role in the occurrence and development of oral squamous cell carcinoma (OSCC). The molecular mechanisms underlying such interaction, however, remain far from clear. Accumulating data have indicated that microRNAs involved in tumor microenvironment, particularly in CAFs, contribute to the activation of fibroblasts and metastasis of cancer cells. Here, we showed that miR‐148a was downregulated in CAFs compared with normal fibroblasts isolated from clinical OSCC tissue. Investigation of miR‐148a function in fibroblasts demonstrated that overexpression of miR‐148a in CAFs significantly impaired the migration and invasion of oral carcinoma cells (SCC‐25) by directly targeting WNT10B. Taken together, these data suggested that miR‐148a might be a novel candidate target for the treatment of OSCC.     

SATB2 overexpression promotes oral squamous cell carcinoma progression by up-regulating NOX4

While atypical expression of special AT-rich sequence-binding protein 2 (SATB2) has been approved associated with tumor progression, metastasis and unfavourable prognosis in various carcinomas. However, in oral squamous cell carcinoma (OSCC), both the expressive state and associated functions of SATB2's are still undefined. Here we show that, in clinical samples from a retrospective cohort of 58 OSCC patients, high expression of SATB2 is associated with poor prognosis of OSCC patients. In this study, we investigated SATB2 is highly expressed in OSCC tissues and cell lines, which can promote OSCC cells' proliferation, migration, invasion and tumor growth. According to sequencing results based on previous literature, we identified NOX4 is a bona fide downstream target of SATB2, when it was knockdown, OSCC's proliferation can be partially suppressed. Furthermore, NOX4 knockdown inhibits tumorigenicity, which can be rescued partially by ectopic expression of SATB2 in HNSCC cell line, and vice versa. Collectively, our findings not only indicate overexpression of SATB2 triggers the proliferative, migratory and invasive mechanisms which are important in the malignant phenotype of OSCC, but also identify NOX4 as the downstream gene for SATB2. These findings indicate that SATB2 may play a key role in OSCC tumorigenicity and may be a future target for the development of new therapeutic regimens.     

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas,Which Contributes to Poor Prognosis

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.