CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.
ABSTRACT: The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ? 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.
Project description:The cyclin-dependent kinase inhibitor 3 (CDKN3) has been perceived as a tumour suppressor. Paradoxically, CDKN3 is often overexpressed in human cancer. It was unclear if CDKN3 overexpression is linked to alternative splicing variants or mutations that produce dominant-negative CDKN3.We analysed CDKN3 expression and its association with patient survival in three cohorts of lung adenocarcinoma. We also examined CDKN3 mutations in the Cancer Genome Atlas (TCGA) and the Moffitt Cancer Center's Total Cancer Care (TCC) projects. CDKN3 transcripts were further analysed in a panel of cell lines and lung adenocarcinoma tissues. CDKN3 mRNA and protein levels in different cell cycle phases were examined.CDKN3 is overexpressed in non small cell lung cancer. High CDKN3 expression is associated with poor overall survival in lung adenocarcinoma. Two CDKN3 transcripts were detected in all samples. These CDKN3 transcripts represent the full length CDKN3 mRNA and a normal transcript lacking exon 2, which encodes an out of frame 23-amino acid peptide with little homology to CDKN3. CDKN3 mutations were found to be very rare. CDKN3 mRNA and protein were elevated during the mitosis phase of cell cycle.CDKN3 overexpression is prognostic of poor overall survival in lung adenocarcinoma. CDKN3 overexpression in lung adenocarcinoma is not attributed to alternative splicing or mutation but is likely due to increased mitotic activity, arguing against CDKN3 as a tumour suppressor.
Project description:The long non-coding RNA (lncRNA) NF-?B interaction lncRNA (NKILA) has been found to exert tumor suppressive effects in numerous types of carcinoma; however, the relationship between NKILA and cervical cancer (CC) remains largely unclear. The present study aimed to investigate the effects of NKILA on the proliferation and metastasis of CC cell lines, in addition to the related molecular mechanisms. Reverse transcription-quantitative PCR was used to detect the expression levels of NKILA in cancer tissues and cell lines. The constructed overexpression vector, pcDNA3.1NKILA, and its corresponding negative control sequence were transfected into CaSki cells and short hairpin RNA targeting NKILA and the corresponding negative control sequence were transfected into C-33A cells. Subsequently, the proliferative, migratory and invasive ability, as well as the process of epithelial-mesenchymal transition (EMT) of C-33 A and CaSki cells were analyzed by performing Cell Counting Kit-8, wound healing, Matrigel invasion and western blot assays, respectively. The expression levels of proteins were detected using western blot analysis. The expression levels of NKILA were decreased in CC tissues and CC cell lines (SiHa, C-33A, CaSki and HeLa) and the downregulation of NKILA expression using shRNA was observed to significantly increase the proliferation of CC cells. Conversely, the upregulation of NKILA inhibited the proliferation of CC cells, in addition to significantly inhibiting the migration and invasion of CaSki cells, whereas the knockdown of NKILA promoted the invasion of C-33A cells. Thus, it was hypothesized that NKILA may inhibit the migration and invasion of CC cells via regulation of EMT processes, which was reflected by the expression of ZO-1, E-cadherin, N-cadherin and Vimentin. Furthermore, the overexpression of NKILA significantly inhibited the activation of NF-?B in CaSki cells, whereas the knockdown of NKILA expression promoted the degradation of inhibitory protein-?B and promoted the transfer of p65 into the nucleus in C-33A cells. In conclusion, the results from the present study suggested that NKILA may be involved in the inhibition of migration and invasion in CC cells through regulating EMT processes, which may be related to its inhibition of NF-?B activation.
Project description:CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.
Project description:In China, esophageal squamous cell carcinoma (ESCC), capable of direct invasion and early metastasis, exhibits high mortality. Identification of the molecular basis driving ESCC progression and development of new diagnostic biomarkers are urgently needed. Cyclin-dependent kinase inhibitor 3 (CDKN3) performs crucial roles in the modulation of tumor development. The present study aimed to explore the functions and underlying mechanism of CDKN3 in regulating ESCC cell proliferation and invasion. The expression levels of CDKN3 in ESCC cells were evaluated by reverse transcription-quantitative PCR. Cell counting kit-8 and colony forming assays were used to evaluate cell viability. Wound-healing assay was performed to explore cell migration. Transwell invasion analysis was conducted to investigate the invasive capacity of ESCC cells. Protein levels were detected by western blot assay. The results demonstrated that the expression of CDKN3 was significantly upregulated in ESCC tissues, as predicted using the UALCAN and Gene Expression Omnibus databases. PCR and western blot assays confirmed that CDKN3 was upregulated in ESCC cell lines. Functional assays revealed that CDKN3 knockdown with small interfering RNA decreased the ability of ESCC cells to proliferate, invade and migrate and suppressed G1/S transition. Further mechanistic analyses demonstrated that CDKN3 promoted cell proliferation and invasion by activating the AKT signaling pathway in ESCC cells. To the best of our knowledge, the present study is the first to identify the functions of CDKN3 in ESCC and provide evidence that CDKN3 regulates tumor progression by activating the AKT signaling pathway. Therefore, CDKN3 may serve as a potential effective therapeutic target for ESCC treatment.
Project description:MicroRNAs are involved in diverse biological processes through regulation of gene expression. The microRNA profile has been shown to be altered in cervical cancer (CC). MiR-16-1 belongs to the miR-16 cluster and has been implicated in various aspects of carcinogenesis including cell proliferation and regulation of apoptosis; however, its function and molecular mechanism in CC is not clear. Cyclin E1 (CCNE1) is a positive regulator of the cell cycle that controls the transition of cells from G1 to S phase. In CC, CCNE1 expression is frequently upregulated, and is an indicator for poor outcome in squamous cell carcinomas (SCCs). Thus, in the present brief communication, we determine whether the CCNE1 gene is regulated by miR-16-1 in CC cells. To identify the downstream cellular target genes for upstream miR-16-1, we silenced endogenous miR-16-1 expression in cell lines derived from CC (C-33 A HPV-, CaSki HPV16+, SiHa HPV16+, and HeLa HPV18+ cells), using siRNAs expressed in plasmids. Using a combined bioinformatic analysis and RT-qPCR, we determined that the CCNE1 gene is targeted by miR-16-1 in CC cells. SiHa, CaSki, and HeLa cells demonstrated an inverse correlation between miR-16-1 expression and CCNE1 mRNA level. Thus, miR-16-1 post-transcriptionally down-regulates CCNE1 gene expression. These results, suggest that miR-16-1 plays a vital role in modulating cell cycle processes in CC.
Project description:Lung cancer is among the major causes of cancer deaths, and the survival rate of lung cancer patients is extremely low. Recent studies have demonstrated that the gene CDKN3 is related to neoplasia, but in the literature severe controversy exists over whether it is involved in cancer progression or, conversely, tumor inhibition. In this study, we investigated the expression of CDKN3 and its association with prognosis in lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) using datasets in Lung Cancer Explorer (LCE; http://qbrc.swmed.edu/lce/). We found that CDKN3 was up-regulated in ADC and SCC compared to normal tissues. We also found that CDKN3 was expressed at a higher level in SCC than in ADC, which was further validated through meta-analysis (coefficient = 2.09, 95% CI = 1.50-2.67, P < 0.0001). In addition, based on meta-analysis for the prognostic value of CDKN3, we found that higher CDKN3 expression was associated with poorer survival outcomes in ADC (HR = 1.65, 95% CI = 1.39-1.96, P < 0.0001), but not in SCC (HR = 1.10, 95% CI = 0.84-1.44, P = 0.494). Our findings indicate that CDKN3 may be a prognostic marker in ADC, though the detailed mechanism is yet to be revealed.
Project description:Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CK? phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.
Project description:Chronic inflammation plays an important role in tumorigenesis of cervical cancer. CD200Fc, a CD200R1 agonist, has been found to have anti-inflammatory effects in autoimmune diseases and neuro-degeneration. However, the anti-inflammatory effect of CD200Fc on cervical cancer has not yet to be completely understood. This study investigated the anti-inflammatory effects and mechanisms of CD200Fc in LPS-induced human SiHa cells and Caski cells. SiHa cells and Caski cells were stimulated with 40 ?g/ml LPS under different concentrations of CD200Fc for 90 min or 12 hours. The mRNA and protein levels of pro-IL-1?, cleaved-IL-1? and NLRP3, as well as the protein level of cleaved caspase-1, were significantly increased in LPS-induced SiHa cells and Caski cells. LPS stimulation did not change ASC and pro-caspase-1 expression. CD200Fc down-regulated protein expression of cleaved caspase-1 and mRNA and protein expression of pro-IL-1?, cleaved-IL-1? and NLRP3. In addition, the protein levels of TLR4, p-P65 and p-I?B, as well as the translocation of P65 to nucleus, were significantly increased in LPS-induced SiHa cells and Caski cells. LPS stimulation did not change t-P65 and t-I?B on protein levels, which were components of TLR-NF-?B pathway. CD200Fc down-regulated protein expression of TLR4, p-P65 and p-I?B and inhibited the translocation of P65 to nucleus in LPS-induced SiHa cells and Caski cells. These results indicated that CD200Fc appeared to suppress the inflammatory activity of TLR4-NF-?B and NLRP3 inflammasome pathway in LPS-induced SiHa cells and Caski cells. It provided novel mechanistic insights into the potential therapeutic uses of CD200Fc for cervical cancer.
Project description:BACKGROUND:MiR-125 has been shown to be involved in a variety of cancers, including cervical cancer (CC). Here, our goal was to explore miR-125 functional role and molecular mechanism in cervical cancer development and progression. METHODS:qRT-PCR was employ to detect miR-125 and VEGF mRNA expression. Western blot was applied for testing protein levels (VEGF, E-cadherin, N-cadherin, vimentin, AKT, p-AKT, PI3K, and p-PI3K). MTT and transwell assays were used for detecting cervical cancer cell progression, including cell viability, migration, and invasion. RESULTS:We observed that miR-125 was downregulated, whereas VEGF was upregulated in cervical cancer tissues and cell lines (CaSki and SiHa). MiR-125 inhibited the proliferation, invasion, and migration by targeting VEGF in cervical cancer. Moreover, miR-125 negatively regulated VEGF expression in cervical cancer tissues. Finally, we demonstrated that miR-520d-5p inhibited the activation of PI3K/AKT signaling pathway. CONCLUSION:In conclusion, the findings demonstrated that miR-125 inhibited cervical cancer progression and development by suppression VEGF and PI3K/AKT signaling pathway.
Project description:Emerging evidence indicates that circRNAs play essential roles in tumorigenesis and development. However, the role of circRNAs in cervical cancer (CC) remains unclear. CircRNA microarrays performed on the immortal cervical cell line H8 and the cervical cancer cell line SiHa were used to identify a circRNA, termed circNRIP1 (hsa_circ_0004771), which was upregulated in SiHa. QRT-PCR confirmed that circNRIP1 was upregulated in CC tissues, where its expression was correlated with lymphovascular space invasion. Besides, both in vitro and in vivo experiments demonstrated that circNRIP1 promotes cell proliferation, migration, and invasion. Additionally, we found that miR-629-3p induced tumor suppression by regulating PTP4A1 and the ERK1/2 pathway. Finally, we confirmed that circNRIP1 exerts its effect, at least partially, by sponging miR-629-3p and thereby regulating the PTP4A1/ERK1/2 pathway. Therefore, circNRIP1 may be useful as a potential prognostic biomarker and therapeutic target in CC patients.