RIPK4 promotes bladder urothelial carcinoma cell aggressiveness by upregulating VEGF-A through the NF-κB pathway.

BACKGROUND: Constitutively activated nuclear factor kappa B (NF-κB) signalling plays vital roles in bladder urothelial carcinoma (BC) progression. We investigate the effect of receptor-interacting protein kinase 4 (RIPK4) on NF-κB activation and BC progression. METHODS: The expression of RIPK4 was examined in 25 cryopreserved paired bladder samples and 112 paraffin BC specimens. In vivo and in vitro assays were performed to validate effect of RIPK4 on NF-κB pathway-mediated BC progression. RESULTS: High expression of RIPK4 was observed in BC tissues and was an independent predictor for poor overall survival. Up or downregulating the expression of RIPK4 enhanced or inhibited, respectively, the migration and invasion of BC cells in vitro and in vivo. Mechanistically, RIPK4 promoted K63-linked polyubiquitination of tumour necrosis factor receptor-associated factor 2 (TRAF2), receptor-interacting protein (RIP) and NF-κB essential modulator (NEMO). RIPK4 also promoted nuclear localisation of NF-κB-p65, and maintained activation of NF-κB substantially, leading to upregulation of VEGF-A, ultimately promoting BC cell aggressiveness. CONCLUSIONS: Our data highlighted the molecular aetiology and clinical significance of RIPK4 in BC: upregulation of RIPK4 contributes to NF-κB activation, and upregulates VEGF-A, and BC progression. Targeting RIPK4 might represent a new therapeutic strategy to improve survival for patients with BC.

MicroRNA-26b Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells by Targeting EphA2.

Sensitizing hepatocellular carcinoma (HCC) cells to irradiation is important to achieve satisfactory therapeutic effect with low-dose radiotherapy. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is a member of the Eph receptor family that constitutes the largest family of tyrosine kinase receptors. EphA2 overexpression is one of the poor prognostic factors in many progressive cancers. Importantly, EphA2 is a potential target of microRNA-26b (miR-26b), and miR-26b expression is down-regulated in several types of cancer. In this study, we measured the expression levels of miR-26b and EphA2 protein in seven human HCC cell lines by quantitative PCR and western blot analysis, respectively. Overall, lower miR-26b expression levels tended to be associated with higher EphA2 levels in HCC cell lines. Among the cell lines examined, 97H HCC cells expressed the lowest level of miR-26b and highest level of EphA2 protein. Thus, using 97H HCC cells, EphA2 mRNA was verified as the target of miR-26b by the luciferase reporter assay. Accordingly, a synthetic miR-26b, miR-26b mimics, was used to mimic the function of endogenous miR-26b. In 97H HCC cells transfected with miR-26b mimics or short-hairpin RNA targeting EphA2 mRNA, expression of EphA2 protein was reduced, which was associated with significantly lower proliferation rate and invasion ability and with higher apoptosis rate in response to low-dose irradiation, compared to control cells. In contrast, 97H HCC cells over-expressing EphA2 showed higher proliferation rate and invasion ability and lower apoptosis rate upon irradiation. These data suggest that miR-26b enhances the radiosensitivity of 97H HCC cells by targeting EphA2 protein.

Retrospective study of hypofractionated stereotactic radiotherapy combined with whole brain radiotherapy for patients with brain metastases.

BACKGROUND AND PURPOSE: To evaluate the clinical outcomes of hypofractionated stereotactic radiotherapy (HFSRT) combined with whole brain radiotherapy (WBRT) in patients with brain metastases (BMs). MATERIALS AND METHODS: From May 2018 to July 2020, 50 patients (111 lesions) received HFSRT (18 Gy/3F) + WBRT (40 Gy/20F). The RECIST 1.1 and RANO-BM criteria were used to evaluate treatment efficacy. Five prognostic indexes (RPA, GPA, SIR, BS-BM, and GGS) were applied. The primary endpoint was intracranial local control (iLC). Secondary endpoints were overall survival (OS) and the safety of treatment. RESULTS: Intracranial objective response rates (iORR) using the RECIST 1.1 and RANO-BM criteria were 62.1% and 58.6%, respectively. The iLC rate was 93.1%, the 6- and 12-month iLC rates were 90.8% and 57.4%, respectively. The median intracranial progression-free survival (iPFS) was not reached (range 0-23 months). The 6-, 12-, and 24-month OS rates were 74.2%, 58.2%, and 22.9%, respectively. The KPS score showed statistical significance in univariate analysis of survival. The 6, 12, and 24 month OS rates for patients with KPS ≥ 70 were 83.8%, 70.5%, and 29.7%, respectively. The median survival time (MST) for all patients and for patients with KPS ≥ 70 were 13.6 and 16.5 months, respectively. Sex, KPS score, and gross tumor volume were significant factors in the multivariate analysis of survival. OS was significantly associated with RPA, SIR, BS-BM, and GGS classes. No acute toxicities of grade 3 or higher were noted. CONCLUSION: HFSRT combined with WBRT is a safe and effective local treatment modality for BM patients.

LncRNA HOTAIR recruits SNAIL to inhibit the transcription of HNF4α and promote the viability, migration, invasion and EMT of colorectal cancer.

Colorectal cancer causes severe burdensome on the health by its high fatality and poor prognosis. Hox transcript antisense intergenic RNA (HOTAIR) was believed closely related with the genesis and development of colorectal cancer, but the regulatory mechanism is still to be investigated. The expression of HOTAIR was analyzed in colorectal cancer using both qRT-PCR and ISH assay. The cell viability, migration, invasion and apoptosis rate were evaluated using MTT, BrdU,Transwell and flow cytometryexperiments. The interaction between HOTAIR and SNAIL was detected using RIP and RNA pull-down. The binding of SNAIL to HNF4α promoter was assessed by ChIP. The cell lines that knock down HOTAIR, SNAIL or overexpress HNF4α were constructed using retroviral vector system. The tumorigenic and metastatic capacity of colorectal cancer cells after knocking down HOTAIR were evaluated based on xenograft assay and liver metastases model. HOTAIR was highly expressed in both tissue and cell lines of colorectal cancer, indicated a regulatory function in colorectal cancer. Knock-down of HOTAIR suppressed cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer cells in vitro, and inhibited the growth and metastasis of colorectal tumor in nude mice. We further found that HOTAIR suppressed HNF4α via recruiting SNAIL, and the overexpression of HNF4α inhibited cell viability, migration, invasion and EMT of colorectal cancer cells. We demonstrated that HOTAIR regulates the level of HNF4α via recruiting SNAIL, knocking down HOTAIR repressed the cell viability and metestasis of colorectal cancer cell line in vitro, and suppressed the tomorgenesis and migration/invasion of colorectal cancer in vivo.

MEG3 Induces Cervical Carcinoma Cells' Apoptosis Through Endoplasmic Reticulum Stress by miR-7-5p/STC1 Axis.

Background: Many patients with advanced cervical cancer (CC) have a poor prognosis and their mortality rank the first among women with malignant tumors. It's essential to explore the molecular mechanism of CC in clinical practice. Long noncoding RNA maternally expressed gene 3 (MEG3) has been reported to downregulate in CC tissues. However, the underlying mechanism of MEG3 in CC remains poorly elaborated. The current study aimed to explore the potential mechanism of MEG3 inducing endoplasmic reticulum stress (ERs)-mediated apoptosis of CC cells. Methods: The expression of MEG3 and miR-7-5p in CC tissues and cell lines was verified by quantitative reverse transcription/polymerase chain reaction (qRT-PCR). The vector of MEG3, miR-7-5p inhibitor, and sh-SCT1 were transfected into CC cell lines, and their expression was tested by qRT-PCR. Flow cytometry was used to detect apoptosis, and ERs-related protein expression was performed by Western blot. The regulatory relationship between MEG3/SCT1 and miR-7-5p was validated by Dual luciferase reporter assay. Results: CC tissues and cell lines showed downregulated MEG3 and STC1, and upregulated miR-7-5p. Overexpression of MEG3 or miR-7-5p inhibition induced ERs-triggered apoptosis of CC cells. In addition, sh-STC1 can reverse the effects of overexpressing MEG3 on CC cell apoptosis. In addition, dual luciferase reporter assay revealed that miR-7-5p can directly target to MEG3 and STC1. Conclusion: MEG3, act as a competing endogenous RNA of miR-7-5p, accelerates ERs-mediated apoptosis of CC cells through regulating SCT1 expression.

SiRNA targeting PFK1 inhibits proliferation and migration and enhances radiosensitivity by suppressing glycolysis in colorectal cancer.

PURPOSE: This study explored the effects of phosphofructokinase-1 (PFK1) on the radiosensitivity of colorectal cancer (CRC) in vivo and in vitro and the underlying mechanisms. METHODS: Tissue samples from 48 patients with rectal cancer who had received neoadjuvant radiotherapy followed by surgery were analyzed. The expression of PFK1 in tissue samples was semi-quantitated by immunohistochemistry, and its relationship with clinicopathological features was analyzed. The effects of PFK1 knockdown on the survival, apoptosis, migration, and radiosensitivity of CRC cells were evaluated. Glycolysis-related indicators were used to examine glycolytic activity. The effects of PFK1 on the radiosensitivity of CRC in vivo were assessed by measuring tumor formation in nude mice. RESULTS: PFK1 was overexpressed in rectal cancer and was higher in radiation-resistant tumors than in radiation-sensitive tumors. SiRNA-induced PFK1 silencing increased apoptosis and inhibited migration and proliferation of CRC cells. Knockdown of PFK1 made the CRC cells sensitive to ionizing radiation in vivo. Oligomycin partially restored the expression of PFK1, enhanced glycolysis, and reversed the enhanced radiosensitivity of CRC cells induced by siRNA-PFK1. Downregulation of PFK1 combined with irradiation inhibited growth of nude mice xenografts, which was related to an increase in apoptosis. CONCLUSIONS: Our study indicates that high expression of PFK1 is negatively correlated with radiosensitivity in CRC and likely accelerates the proliferation and migration of CRC cells. Downregulation of PFK1 may enhance the radiosensitivity of CRC cells in vivo and in vitro by inhibiting glycolysis.

Colorectal cancer in cases of multiple primary cancers: Clinical features of 59 cases and point mutation analyses.

The present study aimed to investigate the occurrence and clinical features of cases of multiple primary cancers including colorectal cancer (MPCC). The medical records of patients with colorectal cancer (CRC) who underwent surgery at the Third Xiangya Hospital of Central South University (Changsha, China) between August 2007 and August 2014 were retrospectively analyzed. Patients with MPCCs were identified and mutation analyses were performed on colon specimens. The results revealed that among 1,311 patients with CRC, 59 had MPCC (including 35 cases of ≥1 CRC with ≥1 other cancer type, and 24 cases with multiple CRCs and no other primary cancers). Foci occurred on the right side of the colon (n=32), in the rectum (n=28), and on the left side of the colon (n=24). MPCCs were synchronous in 24 patients, metachronous in 32 patients, and both in 3 patients. Age of onset and presence of polyps were identified as significantly different between MPCC and CRC overall (P<0.05); however, sex or adenoma incidence were not observed to differ significantly between groups. Mutation incidence rates in 26 specimens were 11.54% for KRAS proto-oncogene GTPase (KRAS) G13D, 3.85% for KRAS Q61R and 3.85% B-Raf proto-oncogene serine/threonine kinase V600E. Mutations of exon 21 of the epithelial growth factor receptor gene, including L858R and L861Q, and of KRAS G12V were not detected. In conclusion, the likelihood of occurrence of MPCC is closely associated with the age of onset and the presence of polyp(s). Routine examination of multiple systems is necessary for patients with CRC to avoid missed diagnosis and misdiagnosis. Further study is required to demonstrate the molecular mechanism of CRC in cases of multiple primary cancers.

miR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2.

OBJECTIVES: Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). MATERIALS AND METHODS: The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. RESULTS: Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. CONCLUSION: These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma.

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9%) and 58 (68.2%) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95% confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95%CI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95%CI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95%CI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95%CI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95%CI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95%CI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9 percent) and 58 (68.2 percent) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95 percent confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95 percentCI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95 percentCI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95 percentCI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95 percentCI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95 percentCI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95 percentCI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.