Long Non-Coding RNA STARD13-AS Suppresses Cell Proliferation And Metastasis In Colorectal Cancer.
ABSTRACT: Background:Dysregulation of long non-coding RNAs (lncRNAs) is closely related with the progression of cancer in humans. The functional and regulatory roles of lncRNAs in colorectal cancer (CRC) are still largely unclear. The purpose of this study is to explore the function of lncRNA STARD13-AS in CRC. Methods:The bioinformatics tool "GEPIA" was used to predict the potential expression of STARD13-AS in CRC. qRT-PCR was used to evaluate the relative expression level of STARD13-AS in CRC cells lines and tissues samples. The functional involvement of STARD13-AS in the CRC cells was assessed using MTT assay, ?ow cytometry, and Transwell assay. The expression levels of cyclin D, cyclin E, E-cadherin, N-cadherin, and vimentin were assessed using Western blot. Results:Bioinformatics prediction and qRT-PCR results showed that STARD13-AS expression was decreased in CRC tissues. Patients with low STARD13-AS expression exhibited distant and lymphatic metastasis as well as enhancement in tumor size. STARD13-AS expression was downregulated in CRC cell lines compared to normal human colon mucosal epithelial cell line NCM460 and STARD13-AS expression in SW620 and LoVo cell lines was lowest. Moreover, we observed that while STARD13-AS overexpression suppressed the cell cycle, proliferation, migration, and invasion, while promoted apoptosis both in LoVo and SW620 cells. In addition, STARD13-AS overexpression inhibited Cyclin E, Cyclin D, N-cadherin and vimentin expression, and promoted E-cadherin expression both in LoVo and SW620 cells. Conclusion:Expression of STARD13-AS suppresses cell proliferation and metastasis in CRC, suggesting that STARD13-AS might act as a potential target for CRC treatment.
Project description:A correlation of TCF12 mRNA overexpression with colorectal cancer (CRC) metastasis was suggested by microarray data and validated by the survey of 120 patients. Thirty-three (27.5%) of the 120 patients showed tumor TCF12 mRNA overexpression and had a higher rate of metastatic occurrence (p = 0.020) and a poorer survival outcome (p = 0.014). Abundant TCF12 levels were also observed in human CRC cell lines such as SW620 and LoVo, but a relatively low level was detected in SW480 cells. Knockdown of TCF12 expression in SW620 and LoVo cells drastically reduced their activities of migration, invasion, and metastasis. Tight cell-cell contact and an increase in E-cadherin but a concomitant decrease in fibronectin were observed in TCF12-knockdown cells. Connexin 26, connexin 43, and gap-junction activity were also increased upon TCF12-knockdown. In contrast, ectopic TCF12 overexpression in SW480 cells facilitated fibronectin expression and cell migration and invasion activities but diminished cellular levels of E-cadherin, connexin 26, connexin 43, and gap junction. A physical association of TCF12 with the E-cadherin promoter was evidenced by chromatin immunoprecipitation assay. TCF12 was tightly correlated with cellular expression of Bmi1 and EZH2 and was co-immunoprecipitable with Bmi1 and EZH2, suggesting that TCF12 transcriptionally suppressed E-cadherin expression via polycomb group-repressive complexes. Clinically, TCF12 mRNA overexpression was also correlated with E-cadherin mRNA down-regulation in the tumor tissues of our 120 patients (p = 0.013). These studies suggested that TCF12 functioned as a transcriptional repressor of E-cadherin and its overexpression was significantly correlated with the occurrence of CRC metastasis.
Project description:microRNA-374a (miR-374a) exhibits oncogenic functions in various tumor types. Here we report that miR-374a suppresses proliferation, invasion, migration and intrahepatic metastasis in colon adenocarcinoma cell lines HCT116 and SW620. Notably, we detected that PI3K/AKT signaling and its downstream cell cycle factors including c-Myc, cyclin D1 (CCND1), CDK4 and epithelial-mesenchymal transition (EMT)-related genes including ZEB1, N-cadherin, Vimentin, Slug, and Snail were all significantly downregulated after miR-374a overexpression. Conversely, cell cycle inhibitors p21 and p27 were upregulated. Expression of E-cadherin was only decreased in HCT116, without any obvious differences observed in SW620 cells. Furthermore, luciferase reporter assays confirmed that miR-374a could directly reduce CCND1. Interestingly, when CCND1 was silenced or overexpressed, levels of pPI3K, pAkt as well as cell cycle and EMT genes were respectively downregulated or upregulated. We examined miR-374a levels by in situ hybridization and its correlation with CCND1 expression in CRC tumor tissues. High miR-374a expression with low level of CCND1 was protective factor in CRC. Together these findings indicate that miR-374a inactivates the PI3K/AKT axis by inhibiting CCND1, suppressing of colon cancer progression.
Project description:MicroRNAs (miRNAs/miRs) are small non-coding RNAs that serve a post-transcriptional regulatory role in eukaryotes. Previous studies have demonstrated that the expression of miR-34a in colorectal cancer (CRC) tissues is decreased compared with that in normal colorectal tissues. However, the role of miR-34a in the invasion and metastasis of CRC remains unclear. In the present study, the levels of miR-34a expression were measured in various CRC cell lines. The cells were transfected with miR-34a mimics or inhibitors in order to assess the proliferation rate, and the colony forming, invasive and migratory abilities. Furthermore, the protein expression levels of vimentin and early growth response protein 1 (EGR1) were examined by western blot analysis. The results revealed that the expression of miR-34a was low in SW620, RKO, LoVo and Caco-2 cell lines and high in the SW480 and SW1116 cell lines. The migration, invasion and proliferation levels of SW480 cells were facilitated by decreasing the expression of miR-34a. Transient transfection with miR-34a mimics in SW620 cells caused a notable decrease in cell migration, invasion and proliferation levels compared with the control group, and a downregulation of vimentin and upregulation of EGR1 protein expression. The present study demonstrated that miR-34a was deregulated in a highly invasive CRC cell lines, and that it may attenuate the migratory, invasive and proliferative capabilities of CRC cells by enhancing the expression of EGR1 and inhibiting that of vimentin. The results of the present study represent important progress towards understanding the mechanisms of CRC recurrence and metastasis.
Project description:Our previous study has demonstrated that knockdown of Grainyhead-like 2(GRHL2) in colorectal cancer (CRC) cells inhibited cell proliferation by targeting ZEB1. This study aimed at researching whether knockdown of GRHL2 promoted CRC progression and metastasis via inducing epithelial-mesenchymal transition (EMT). GRHL2-upregulated SW-620/GRHL2+ and GRHL2-knockdown HCT116/GRHL2-KD, HT29/GRHL2-KD cells and their control cells were generated. The morphological changes after overexpression and knockdown GRHL2 were observed. qRT-PCR, Western blotting, and Immunofluorescence were used to detect EMT markers: E-cadherin, Vimentin, p-catein, ZO-1 and ZEB1 expression. Then, sh-ZEB1 was transfected to GRHL2 knockdown cells to research the relationship between GRHL2 and ZEB1. Transwell and wound healing assays were further performed to detect the impact of GRHL2 on invasion and migration in vitro. CRC cells were injected into mice tail vein to verify the impact of GRHL2 on CRC metastasis. Morphological change of mesenchymal-epithelial transition (MET) could be observed in SW620/GRHL2+ cell. The expression of epithelial markers: E-cadherin, ?-catenin, ZO-1 were up-regulated, while mesenchymal markers: Vimentin was decreased. Meanwhile, opposite EMT morphological change could be observed in HCT116/GRHL2-KD cell, accompanied by reverse change of E-cadherin, ?-catenin, ZO-1, and Vimentin. The expression level of GRHL2 and ZEB1 was found negative in both SW620/GRHL2+ and HCT116/GRHL2-KD cells. Knockdown of ZEB1 by siRNA in HCT116/GRHL2-KD and HT29/GRHL2-KD could upregulate expression of E-cadherin and GRHL2. GRHL2 knockdown also promoted migration, invasion in vitro and CRC metastasis in mice model. In conclusion, GRHL2/ZEB1 axis inhibits CRC progression and metastasis via oppressing EMT.
Project description:The progression of colorectal carcinoma (CRC) to invasive and metastatic disease may involve localized occurrences of epithelial-mesenchymal transition (EMT). However, mechanisms of the EMT process in CRC progression are not fully understood. We previously showed that knockdown of signal transducer and activator of transcription 3 (STAT3) up-regulated E-cadherin (a key component in EMT progression) in CRC. In this study, we examined the roles of STAT3 in CRC EMT and ZEB1, an EMT inducer, in STAT3-induced down-regulation of E-cadherin. Knockdown of STAT3 significantly increased E-cadherin and decreased N-cadherin and vimentin expressions in highly invasive LoVo CRC cells. Meanwhile, overexpression of STAT3 significantly reduced E-cadherin and enhanced N-cadherin and vimentin expressions in weakly invasive SW1116 CRC cells. Activation of STAT3 significantly increased CRC cell invasiveness and resistance to apoptosis. Knockdown of STAT3 dramatically enhanced chemosensitivity of CRC cells to fluorouracil. STAT3 regulated ZEB1 expression in CRC cells, and the STAT3-induced decrease in E-cadherin and cell invasion depended on activation of ZEB1 in CRC cells. Additionally, pSTAT3(Tyr-705) and ZEB1 expressions were significantly correlated with TNM (tumor, lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT3 may directly mediate EMT progression and regulate ZEB1 expression in CRC. ZEB1 may participate in STAT3-induced cell invasion and E-cadherin down-regulation in CRC cells. The expressions of pSTAT3(Tyr-705) and ZEB1 may be positively associated with CRC metastasis. Our data may provide potential targets to prevent and/or treat CRC invasion and metastasis.
Project description:Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ? 0.001-0.003) and suppressed invasiveness by ?3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of ?-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.
Project description:Background:An increasing number of studies demonstrate that long non-coding RNAs (lncRNAs) are regulators in cancer biology. Nevertheless, the expression and mechanism of LINC01089 in colorectal cancer (CRC) remain unclear. Methods:Quantitative real-time polymerase chain reaction (qRT-PCR) was taken to investigate the expression levels of LINC01089 and miR-27b-3p in CRC tissues and cells. MTT method and transwell test were employed to assess the proliferation and invasion of CRC cells, respectively. Dual-luciferase activity reporter assay, RNA immunoprecipitation assay, Pearson's correlation analysis, and Western blot were performed to investigate the regulatory mechanism of LINC01089/miR-27b-3p/HOXA10 axis in CRC. Results:LINC01089 was down-regulated in CRC tissues and cell lines. LINC01089 overexpression impeded the proliferation and invasion of SW620 and LoVo cells, whereas LINC01089 knockdown increased the malignancy of SW480 and HT29 cells. Moreover, LINC01089 directly interacted with miR-27b-3p to repressed its expression and indirectly promoted the expression of HOXA10. Conclusion:LINC01089 impedes the proliferation and invasion of colorectal cancer cells by adsorbing miR-27b-3p and up-regulating the expression of HOXA10.
Project description:Purpose:STARD13 is regulated by various miRNAs. However, there are relatively few reports describing the relationship between miRNAs and STARD13 in pancreatic cancer. Therefore, the aim of this study was to explore the relationship between miRNA and STARD13 in pancreatic cancer. Patients and Methods:By analyzing the data from Gene Expression Omnibus (GEO) database, the relationship between STARD13 expression and pancreatic cancer was explored. Then, through sequence alignment, the sequence complementary to miR-887-3p in the 3'UTR of STARD13 mRNA was found, mutated and cloned. Dual-luciferase reporter assay was used to test the relationship between STARD13 and miR-887-3p. Pancreatic cancer tumor tissue and its adjacent tissues collected, and the expression of STARD13 and miR-887-3p in pancreatic cancer tissues was analyzed by RT-qPCR. After, miR-887-3p and its inhibitor were transfected into PANC-1 cells to further confirm the regulatory relationship between miR-887-3 and STARD13 by RT-qPCR, and CCK-8, colony formation assays, cell cycle analysis, apoptosis detection and transwell analysis were used to detect changes of proliferation, apoptosis, migration and invasion in PANC-1 cells. Finally, through in vivo experiments, the effect of miR-887-3p on tumor growth was researched. Results:We found that STARD13 expression is lower in pancreatic cancer tissues, with the level of miR-887-3p being higher in these tissues. Pancreatic cancer patients with particularly low levels of STARD13 presented with a poor prognosis. MiR-887-3p negatively regulates the expression of STARD13. Increasing levels of miR-887-3p decreased the expression of STARD13, which promoted the proliferation, cell cycle process, cell migration and invasion, and inhibited the apoptosis of pancreatic cancer cells. Inhibition of miR-887-3p in SCID mice could inhibit tumor growth and promote tumor cell apoptosis. Conclusion:In conclusion, STARD13 is negatively regulated by miR-887-3p in pancreatic cancer. MiR-877-3p may act to promote cancer progression, and as such, it is a viable target for intervention and diagnostic development.
Project description:The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT), are involved in the progression, metastasis, and resistance of colorectal cancer (CRC) to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a-15k) and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j) significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21) to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, ?-catenin, c-Myc) and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro, 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.
Project description:Acyl-CoA synthetase 5 (ACS5) has been reported to be associated with the development of various cancers, but the role of it in colorectal cancer (CRC) is not well understood. The present study aimed to explore the potential role of ACS5 in the development and progression of CRC.ACS5 expression in CRC tissues and CRC cell lines was examined, and its clinical significance was analyzed. The role of ACS5 in cell proliferation, apoptosis, and invasion was examined in vitro.We found that ACS5 expression was upregulated in CRC cells and CRC tissues and that high ACS5 expression was more frequent in CRC patients with excess muscular layer and with poor tumor differentiation. Furthermore, knockdown of ACS5 in HT29 and SW480 cells significantly dampened cell proliferation, induced cell apoptosis, and reduced cell migration and invasion. In contrast, the ectopic overexpression of ACS5 in LOVO and SW620 cells remarkably promoted cell proliferation, inhibited cell apoptosis, and enhanced cell migration and invasion. Enhanced cell growth and invasion ability mediated by the gain of ACS5 expression were associated with downregulation of caspase-3 and E-cadherin and upregulation of survivin and CD44.Our data demonstrate that ACS5 can promote the growth and invasion of CRC cells and provide a potential target for CRC gene therapy.