LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells

Object

This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells.

Proteomic analysis of Tiam1-mediated metastasis in colorectal cancer

Tiam1 (T lymphoma invasion and metastasis 1), a guanine nucleotide exchange factor that activates Rac, was recently identified as a novel colorectal cancer metastasis-related gene. To better understand the mechanism underlying Tiam1-mediated metastasis, we applied two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis to identify differentially expressed proteins between Tiam1 transfected and mock transfected colorectal cancer HT29 cells. Eleven differentially expressed proteins were identified and further validated by Western blot and/or real-time PCR. The results revealed that Tiam1 transfection in colorectal cancer cells could upregulate the expression of Fascin-1, heat shock protein 27 (HSP27), high-mobility group box 1 (HMGB1), glutathione S-transferase omega 1 (GSTO1) and downregulate the expression of annexin IV. These differentially expressed proteins may be directly or indirectly regulated by Tiam1 and be helpful in studying mechanisms that lead to the function of Tiam1. These results give some clues to elucidate the mechanism of Tiam1-mediated metastasis for colorectal cancer.     

Pseudogene HSPB1P1 contributes to renal cell carcinoma proliferation and metastasis by targeting miR-296-5p to regulate HMGA1 expression

With the aid of next-generation sequencing technology, pseudogenes have been widely recognized as functional regulators in the development and progression of certain diseases, especially cancer. Our present study aimed to investigate the functions and molecular mechanisms of HSPB1-associated protein 1 pseudogene 1 (HSPB1P1) in renal cell carcinoma (RCC). HSPB1P1 expression at the mRNA levels was determined by quantitative real-time polymerase chain reaction, and its clinical significance was assessed. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were detected by transwell assays. The location of HSPB1P1 in RCC cells was detected by subcellular distribution analysis. The direct relationship between HSPB1P1 and miR-296-5p/HMGA1 axis was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results identify the elevated expression of HSPB1P1 in RCC tissues and cell lines, which predicted advanced progression and poor prognosis in patients with RCC. Knockdown of HSPB1P1 suppressed cell proliferation, migration, and invasion, and reversed epithelial–mesenchymal transition process in RCC. HSPB1P1 was mostly enriched in the cytoplasm and functioned as a miRNA sponge for miR-296-5p and then regulated high-mobility group A1 expression. In conclusion, our study indicated that HSPB1P1 contributed to RCC progression by targeting the miR-296-5p/HMGA1 axis, and should be considered as a promising biomarker and therapeutic target for clinical applications.     

Suppression of TWIST1 enhances the sensitivity of colon cancer cells to 5-fluorouracil

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU.     

Inhibition of KHSRP sensitizes colorectal cancer to 5-fluoruracil through miR-501-5p-mediated ERRFI1 mRNA degradation

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3′-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.     

Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.     

Identification of signature genes associated with 5-fluorouracil-resistance in human gastric cancer cells

A comparative cDNA microarray analysis was carried out using human gastric cancer cells and their derivative cells made resistant to 5-fluorouracil. Three genes, SRP72, DNA primase, and caspase-6, were identified that were transiently induced by 5-fluorouracil and also overexpressed in 5-fluorouracil-resistant gastric cancer cells.     

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.     

Identification of potential genes/proteins regulated by Tiam1 in colorectal cancer by microarray analysis and proteome analysis

Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1), a guanine nucleotide exchange factor that activates Rac, is a colorectal cancer metastasis-related gene. In this study, we aimed to better understand the mechanism underlying Tiam1-mediated metastasis. We applied gene microarray and proteome analysis and compared expression of genes and proteins in a stable Tiam1-silencing colorectal cancer cell line and in a control cell line. Our analysis identified three genes, high-mobility group box1 (HMGB1), annexin IV (ANXA4) and phosphoglycerate mutase 1 (PGAM1) that were associated with Tiam1. Analysis of these proteins, which may be directly or indirectly regulated by Tiam1, may provide insight into the role and mechanism of Tiam1 in colorectal cancer metastasis.     

A tRF-5a fragment that regulates radiation resistance of colorectal cancer cells by targeting MKNK1

Radiotherapy serves as a crucial strategy in the treatment of colorectal cancer (CRC). However, its efficacy is often hindered by the challenge of radiation resistance. Although the literature suggests that some tRNA-derived small RNAs (tsRNAs) are associated with various cancers, studies reporting the relationship of tsRNAs with cancer cell radiosensitivity have not been published yet. In our study, we utilized tsRNAs sequencing to predict differentially expressed tsRNAs in two CRC cells and their radioresistant cells, and 10 tsRNAs with significant differences in expression were validated by qPCR. The target genes of tRF-16-7X9PN5D were predicted and verified by the bioinformatics, dual-luciferase reporter gene assay and western blotting analyses. Wound healing, colony formation, transwell invasion and CCK-8 assays were performed to detect the effects of tRF-16-7X9PN5D on cell function and radiosensitivity. Western blotting evaluated the relationship between tRF-16-7X9PN5D and the MKNK-eIF4E axis. Our findings demonstrated that tRF-16-7X9PN5D expression was substantially downregulated in radioresistant CRC cells. Furthermore, tRF-16-7X9PN5D could promote CRC cells' ability to proliferate, migrate, invade and obtain radiation resistance by targeting MKNK1. Finally, tRF-16-7X9PN5D could regulate eIF4E phosphorylation via MKNK1. This investigation indicated that tRF-16-7X9PN5D has an essential regulatory role in the radiation resistance of CRC by directly targeting MKNK1, and may be a new pathway for regulating the CRC radiosensitivity.     

AEG-1对大肠癌进展的影响

目的研究AEG-1在大肠癌组织和细胞中的表达,探讨AEG-1通过调控上皮间质转化和耐药参与大肠癌的进展。方法采用qRT-PCR检测AEG-1在大肠癌组织和细胞中的表达,统计AEG-1对大肠癌患者生存率的影响,分析其在不同癌症分期患者中的表达差异,并分析AEG-1表达量与大肠癌诊断敏感性的关系。采用体外实验将si-NC、pc-DNA-NC、si-AEG-1、pc-DNA-AEG-1转染到大肠癌SW116和LOVO细胞中,然后通过qRT-PCR检测转染效率以及AEG-1在两细胞系中的表达情况。采用CCK-8和克隆形成实验检测AEG-1对大肠癌细胞增殖的影响;采用流式细胞术检测转染后上皮间质转化和耐药情况的变化;采用Western blotting检测转染后上皮间质转化和耐药相关蛋白N-cadherin、E-cadherin、MRP的变化情况。结果 77例大肠癌患者组织中AEG-1表达水平明显高于对照组。浸润T3+T4期的患者中AEG-1的表达水平高于浸润T1+T2期患者。Ⅲ+Ⅳ期患者中AEG-1的表达水平高于Ⅰ+Ⅱ期。AEG-1高表达组患者OS时间明显低于低表达组。AEG-1表达量与大肠癌诊断敏感性之间呈显著正相关。在LOVO细胞系中,降低AEG-1表达后其细胞活力、侵袭力明显降低,同时间质细胞标志蛋白N-cadherin、上皮细胞标志蛋白E-cadherin、多药耐药相关蛋白MRP表达量降低。在SW116细胞系中,过表达AEG-1后上皮间质转化、耐药相关蛋白表达量显著升高。结论 AEG-1在大肠癌组织和细胞中的表达量明显上升,AEG-1通过调控上皮间质转化和耐药参与大肠癌的发生发展,为大肠癌的治疗提供了新的理论依据和新的靶点。     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

miR-129 promotes apoptosis and enhances chemosensitivity to 5-fluorouracil in colorectal cancer

Resistance to fluoropyrimidine-based chemotherapy is the major reason for the failure of advanced colorectal cancer (CRC) treatment. The lack of ability of tumor cells to undergo apoptosis after genotoxic stress is the key contributor to this intrinsic mechanism. Mounting evidence has demonstrated that non-coding microRNAs (miRNAs) are crucial regulators of gene expression, in particular, under acute genotoxic stress. However, there is still limited knowledge about the role of miRNAs in apoptosis. In this study, we discovered a novel mechanism mediated by microRNA-129 (miR-129) to trigger apoptosis by suppressing a key anti-apoptotic protein, B-cell lymphoma 2 (BCL2). Ectopic expression of miR-129 promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest in CRC cells. The intrinsic apoptotic pathway triggered by miR-129 was activated by cleavage of caspase-9 and caspase-3. The expression of miR-129 was significantly downregulated in CRC tissue specimens compared with the paired normal control samples. More importantly, we demonstrated that miR-129 enhanced the cytotoxic effect of 5-fluorouracil both in vitro and in vivo. These results suggest that miR-129 has a unique potential as a tumor suppressor and a novel candidate for developing miR-129-based therapeutic strategies in CRC.     

Comprehensive analysis of pseudogene HSPB1P1 and its potential roles in hepatocellular carcinoma

The incidence and mortality rate of hepatocellular carcinoma (HCC) nowadays is still at high levels. The regulatory roles of pseudogene in cancers have been gradually recognized in recent years. However, comprehensive investigation of abnormally expressed pseudogene and related mechanisms in HCC remains lacking. GSE124535 dataset was used to identify differentially expressed pseudogenes in HCC tissues compared with normal tissues. Prognostic value of these differentially expressed pseudogenes was analyzed at GEPIA. StarBase used to analyze microRNAs (miRNAs) can bind with pseudogene, while the targets for these miRNAs were analyzed at miRTarBase. Protein–protein interaction (PPI) network was then established for miRNA targets, after that hub genes were selected. Expression correlation of pseudogene and hub genes was analyzed at StarBase. In total, 16 upregulated and 17 downregulated pseudogenes were identified. Pseudogene HSPB1P1 was identified abnormally expressed in 20 types of human cancers and could be used as an indicator for poorer overall survival of patients with HCC. Functional analyses showed that HSPB1P1 was strongly correlated with signaling pathways related to cancer progression. Further studied revealed that HSPB1P1 could direct regulate the EZH2 expression in HCC. In summary, our study indicated that HSPB1P1 was a predictor for poorer overall survival of patients with HCC and may be potential therapeutic target against HCC.     

Clinicopathologic significance of ERCC1, thymidylate synthase and glutathione S-transferase P1 expression for advanced gastric cancer patients receiving adjuvant 5-FU and cisplatin chemotherapy

The objective of this study was to determine whether the expressions of the excision cross-complementing (ERCC1), thymidylate synthase (TS) and glutathione S-transferase P1 (GSTP1) are predictive of clinical outcomes in advanced gastric cancer (AGC) patients receiving treatment with adjuvant 5-fluorouracil (5-FU) and cisplatin (FP) chemotherapy. One hundred forty nine patients were included in this study. ERCC1 and GSTP1 expression was correlated significantly with tumor size (p?=?0.040, p?=?0.018, respectively). Stage and positive lymph node ratio were associated independently with disease free survival (DFS) and overall survival (OS). Both ERCC1 and GSTP1 expression had a significant impact on OS (hazard ratio?=?0.069, p?=?0.021). TS expression was not related to DFS and OS.     

Circ-ERBB2 knockdown sensitized colorectal cancer cells to 5-FU via miR-181a-5p/PTEN/Akt pathway

Colorectal cancer (CRC) is the fourth most deadly cancer worldwide, drug resistance impedes treatment of CRC. It is still urgent to find new molecular targets to improve the sensitivity of chemotherapeutic drugs. In this study, circ-ERBB2 was upregulated in CRC cells. Upregulation of circ-ERBB2 promoted CRC cells proliferation and clone formation, but inhibited apoptosis. We identified miR-181a-5p as circ-ERBB2's target. The effect of miR-181a-5p on CRC cells was contrary to circ-ERBB2, miR-181a-5p downregulation abolished the function of circ-ERBB2 silencing in CRC cells. In addition, phosphatase and tensin homolog (PTEN) was verified as miR-181a-5p's downstream target, circ-ERBB2 activates the Akt pathway and inhibits cell apoptosis through modulating miR-181a-5p/PTEN. Circ-ERBB2 silencing significantly reduced CRC cell resistance to 5-FU. miR-181a-5p downregulation abolished the role of circ-ERBB2 knockdown in CRC cell resistance to 5-FU. In conclusion, upregulation of circ-ERBB2 promoted the malignancy of CRC and reduced CRC cell resistance to 5-FU. Besides, additional mechanism study provided a novel regulatory pathways that circ-ERBB2 knockdown promoted CRC cell sensitivity to 5-FU by regulating miR-181a-5p/PTEN/Akt pathway. This research indicated that circ-ERBB2 may be a valuable biomarker for the diagnosis and treatment of CRC.     

MiR-153-5p promotes sensibility of colorectal cancer cells to oxaliplatin via targeting Bcl-2-mediated autophagy pathway

ABSTRACT

Oxaliplatin (L-OHP) is one of the effective chemotherapeutic drugs for colorectal cancer (CRC). Further investigation into the molecular mechanism of chemoresistance could improve outcomes for patients with colorectal cancer. Recently, microRNAs have been reported as a key in drug resistance of tumors. In this study, we aimed to investigate the effects of miR-153-5p in L-OHP-resistant CRC cells, and its underlying mechanism. Downregulation of miR-153-5p was observed in CRC cells, while upregulation of miR-153-5p enhances the chemosensitivity of CRC/L-OHP cells. The autophagy of CRC/L-OHP cells was markedly increased after exposure to L-OHP but abolished by the upregulation of miR-153-5p. Dual-luciferase reporter assays validated that Bcl-2 was a direct target of miR-153-5p. In conclusion, our data suggested that miR-153-5p was a mediator of cisplatin resistance in colorectal cancer by affecting Bcl-2-mediated autophagy, indicating a new therapeutic target for CRC treatment.