MicroRNA miR-324-3p induces promoter-mediated expression of RelA gene.

MicroRNAs (miRNAs) are known to repress translation by binding to the 3'UTRs of mRNAs. Using bioinformatics, we recently reported that several miRNAs also have target sites in DNA particularly in the promoters of the protein-coding genes. To understand the functional significance of this phenomenon, we tested the effects of miR-324-3p binding to RelA promoter. In PC12 cells, co-transfection with premiR-324-3p induced a RelA promoter plasmid in a dose-dependent manner and this effect was lost when the miR-324-3p binding site in the promoter was mutated. PremiR-324-3p transfection also significantly induced the endogenous RelA mRNA and protein expression in PC12 cells. Furthermore, transfection with premiR-324-3p increased the levels of cleaved caspase-3 which is a marker of apoptosis. Importantly, the miR-324-3p effects were Ago2 mediated as Ago2 knockdown prevented RelA expression and cleavage of caspase-3. Thus, our studies show that miRNA-mediated transcriptional activation can be seen in PC12 cells which are neural in origin

Induction of endogenous RelA by miR-324-3p.

<p>When PC12 cells were transfected with 150 nM of premiR-324-3p, there was a significant induction of RelA mRNA expression compared to control premiR treated group at 3 days after transfection (A). Ago2 siRNA treatment prevented the RelA induction by miR-324-3p. The house-keeping control 18S rRNA expression was not changed by premiR-324-3p transfection or Ago2 knockdown (A). PremiR-324-3p transfection also led to significant increase in the RelA protein levels compared to control miR treated or vehicle treated control groups (B). Treatment with Ago2 siRNA prevented the premiR-324-3p mediated increase in RelA protein levels (B). The levels of β-actin used as a loading control were not affected by premiR-324-3p or Ago2 siRNA (B). The efficiency of Ago2 siRNA to knockdown Ago2 protein levels in PC12 cells in the presence and absence of premiR-324-3p and control miR was confirmed by Western blotting. The Ago2 siRNA knocked-down Ago2 protein levels by >85% in all the 3 groups (treated with vehicle or control miR or premiR-324-3p) compared to vehicle treated group (C). PremiR-324-3p transfection induced significant activation of caspase-3 as measured by cleaved caspase-3 levels in PC12 cells which was prevented by Ago2 knockdown (D). Bars represent mean ± SD of n  =  4/group of triplicate determinations. * p<0.05 compared with the control siRNA group (Student’s t-test).</p

miR-324-3p induced RelA promoter.

<p>A miR-324-3p binding sits is present at nucleotides 45 to 66 upstream to RelA transcription start site (TSS) (A). Three bases (shown in bold) in the miR-324-3p binding site were mutated in the RelA promoter to create a mutant promoter vector (A). The RelA promoter vector showed significant dose-dependent induction when cotransfected with premiR-324-3p (B). The RelA mutant vector showed no induction by premiR-324-3p (B). Bars represent mean ± SD (n  =  4/group). Each assay was conducted in triplicate. *p<0.05 and **p<0.01 compared to the respective mutant group (Student’s t-test).</p

The impact of HIPEC vs. EPIC for the treatment of mucinous appendiceal carcinoma: a study from the US HIPEC collaborative

Introduction Mucinous appendiceal carcinoma is a rare malignancy that commonly spreads to the peritoneum leading to peritoneal metastases. Complete cytoreduction with perioperative intraperitoneal chemotherapy (PIC) is the mainstay of treatment, administered as either hyperthermic intra peritoneal chemotherapy (HIPEC) or early post-operative intraperitoneal chemotherapy (EPIC). Our goal was to assess the perioperative and long term survival outcomes associated with these two PIC methods. Materials and methods Patients with mucinous appendiceal carcinoma were identified in the US HIPEC Collaborative database from 12 academic institutions. Patient demographics, clinical characteristics, and survival outcomes were compared among patients who underwent HIPEC vs. EPIC with inverse probability weighting (IPW) used for adjustment. Results Among 921 patients with mucinous appendiceal carcinoma, 9% underwent EPIC while 91% underwent HIPEC. There was no difference in Grade III–V complications between the two groups (18.5% for HIPEC vs. 15.0% for EPIC, p=.43) though patients who underwent HIPEC had higher rates of readmissions (21.2% vs. 8.8%, p<.01). Additionally, PIC method was not an independent predictor for overall survival (OS) or recurrence-free survival (RFS) after adjustment on multivariable analysis. Conclusions Among patients with mucinous appendiceal carcinoma, both EPIC and HIPEC appear to be associated with similar perioperative and long-term outcomes

Comparison of open and closed hyperthermic intraperitoneal chemotherapy: Results from the United States hyperthermic intraperitoneal chemotherapy collaborative.

BackgroundCytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal carcinomatosis can be performed in two ways: Open or closed abdominal technique.AimTo evaluate the impact of HIPEC method on post-operative and long-term survival outcomes.MethodsPatients undergoing CRS with HIPEC from 2000-2017 were identified in the United States HIPEC collaborative database. Post-operative, recurrence, and overall survival outcomes were compared between those who received open vs closed HIPEC.ResultsOf the 1812 patients undergoing curative-intent CRS and HIPEC, 372 (21%) patients underwent open HIPEC and 1440 (79%) underwent closed HIPEC. There was no difference in re-operation or severe complications between the two groups. Closed HIPEC had higher rates of 90-d readmission while open HIPEC had a higher rate of 90-d mortalities. On multi-variable analysis, closed HIPEC technique was not a significant predictor for overall survival (hazards ratio: 0.75, 95% confidence interval: 0.51-1.10, P = 0.14) or recurrence-free survival (hazards ratio: 1.39, 95% confidence interval: 1.00-1.93, P = 0.05) in the entire cohort. These findings remained consistent in the appendiceal and the colorectal subgroups.ConclusionIn this multi-institutional analysis, the HIPEC method was not independently associated with relevant post-operative or long-term outcomes. HIPEC technique may be left to the discretion of the operating surgeon

A novel preoperative risk score to optimize patient selection for performing concomitant liver resection with cytoreductive surgery/HIPEC

BackgroundWhile parenchymal hepatic metastases were previously considered a contraindication to cytoreductive surgery (CRS) and heated intraperitoneal chemotherapy (HIPEC), liver resection (LR) is increasingly performed with CRS/HIPEC.MethodsPatients from the US HIPEC Collaborative (2000-2017) with invasive appendiceal or colorectal adenocarcinoma undergoing primary, curative intent CRS/HIPEC with CC0-1 resection were included. LR was defined as a formal parenchymal resection. Primary endpoints were postoperative complications and overall survival (OS).ResultsA total of 658 patients were included. About 83 (15%) underwent LR of colorectal (58%) or invasive appendiceal (42%) metastases. LR patients had more complications (81% vs. 60%; p = .001), greater number of complications (2.3 vs. 1.5; p &lt; .001) per patient and required more reoperations (22%&nbsp;vs. 11%; p = .007) and readmissions (39% vs. 25%; p = .014) than non-LR patients. LR patients had decreased OS&nbsp;(2-year OS 62% vs. 79%, p &lt; .001), even when accounting for peritoneal carcinomatosis index and histology type. Preoperative factors associated with decreased OS on multivariable analysis in LR patients included age &lt; 60 years&nbsp;(HR, 3.61; 95% CI, 1.10-11.81), colorectal histology (HR, 3.84; 95% CI, 1.69-12.65), and multiple liver tumors (HR, 3.45; 95% CI, 1.21-9.85) (all p &lt; .05). When assigning one point for each factor, there was an incremental decrease in 2-year survival as the risk score increased from 0 to 3 (0: 100%; 1: 91%; 2: 58%; 3: 0%).ConclusionsAs CRS/HIPEC + LR has become more common, we created a simple risk score to stratify patients considered for CRS/HIPEC + LR. These data aid in striking the balance between an increased perioperative complication profile with the potential for improvement in OS

Impact of Neoadjuvant Chemotherapy on the Outcomes of Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for Colorectal Peritoneal Metastases: A Multi-Institutional Retrospective Review.

Cytoreductive surgery (CRS) with or without hyperthermic intraperitoneal chemotherapy (HIPEC) is associated with improved survival for patients with colorectal peritoneal metastases (CR-PM). However, the role of neoadjuvant chemotherapy (NAC) prior to CRS-HIPEC is poorly understood. A retrospective review of adult patients with CR-PM who underwent CRS+/-HIPEC from 2000-2017 was performed. Among 298 patients who underwent CRS+/-HIPEC, 196 (65.8%) received NAC while 102 (34.2%) underwent surgery first (SF). Patients who received NAC had lower peritoneal cancer index score (12.1 + 7.9 vs. 14.3 + 8.5, p = 0.034). There was no significant difference in grade III/IV complications (22.4% vs. 16.7%, p = 0.650), readmission (32.3% vs. 23.5%, p = 0.114), or 30-day mortality (1.5% vs. 2.9%, p = 0.411) between groups. NAC patients experienced longer overall survival (OS) (median 32.7 vs. 22.0 months, p = 0.044) but similar recurrence-free survival (RFS) (median 13.8 vs. 13.0 months, p = 0.456). After controlling for confounding factors, NAC was not independently associated with improved OS (OR 0.80) or RFS (OR 1.04). Among patients who underwent CRS+/-HIPEC for CR-PM, the use of NAC was associated with improved OS that did not persist on multivariable analysis. However, NAC prior to CRS+/-HIPEC was a safe and feasible strategy for CR-PM, which may aid in the appropriate selection of patients for aggressive cytoreductive surgery