ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Synergistic Effect of Rhamnolipids and Inoculation on the Bioremediation of Petroleum-Contaminated Soils by Bacterial Consortia.

Crude oil is a serious soil pollutant, requiring large-scale remediation efforts. Bacterial consortia in combination with rhamnolipids can be an effective bioremediation method. However, the underlying mechanisms and associated changes in soil bacterial composition remain uncharacterized. Therefore, this study sought to evaluate the effectiveness of rhamnolipids in petroleum hydrocarbon removal, and the associated bacterial community dynamics during bioremediation of petroleum-contaminated soils. Contaminated soils were subjected to natural attenuation, bioremediation with rhamnolipids, bioremediation with bacterial consortia, or bioremediation with bacterial consortia supplemented with rhamnolipids (BMR). High-throughput sequencing of bacterial sample partial 16S rRNA sequences was performed. Additionally, the n-alkanes and aromatic fractions were analyzed by gas chromatography-mass spectroscopy. The results showed that rhamnolipid supplementation increased the rate and extent of total petroleum hydrocarbon biodegradation to a maximum of 81% within 35 days. Further, phylogenetic analysis revealed that the bacterial community was composed of 14 phylotypes (similarity level = 97%). Actinobacteria and Proteobacteria were the two core phyla in all samples, accounting for 63-89%, but Proteobacteria was the most dominant phylum in the BMR sample (~ 53%). Among the top 20 genera, Pseudomonas, Pseudoxanthomonas, Cavicella, Mycobacterium, Rhizobium, and Acinetobacter were more abundant in BMR samples compared to other samples. Predicted functional profiles revealed that rhamnolipid addition also induced changes in gene abundance related to hydrocarbon metabolic pathways. This study provided comprehensive insights into the synergistic effect of rhamnolipids and bacterial consortia for altering bacterial populations and specific functional traits, which may serve to improve bacteria-mediated petroleum hydrocarbon biodegradation in contaminated soils.