Estimating Efficacy of Conversion Therapy on Patients with Initially Unresectable Colorectal Cancer Liver Metastases by using MRI: Development of a Predictive Score.

RATIONALE AND OBJECTIVES: The conversion success rate (CSR) has crucial implication for clinical outcomes of initially unresectable colorectal liver metastases (CRLM) following conversion therapy. This study aimed to develop a simple predictive scoring model for identifying CSR according to baseline magnetic resonance imaging (MRI) features, and confirm its performance and prognostic significance in a validation cohort. METHODS: A total of 155 consecutive patients with initially unresectable CRLM were retrospectively reviewed in the study. A simple MRI-based predictive scoring model for identifying CSR was developed in the development cohort (n = 104) by using multivariable logistic regression analyzes. The diagnostic performance was evaluated for the predictive score. Thereafter, patients in the validation cohort (n = 51) were stratified into groups with predicted high CSR or low CSR according to the score. The progression-free survival (PFS) and overall survival (OS) were compared between two groups using the log-rank test. RESULTS: The predictive score of CSR, named mrNISE, incorporated the number of CRLM ≥ 10, the largest size ≥ 50 mm, poorly defined tumor-liver interface, and peritumoral enhancement. The AUC of the mrNISE score was 0.845 for the development cohort and 0.776 for the validation cohort. According to the score, patients with predicted high CSR had better PFS and OS than those with low CSR in both development and validation cohorts. CONCLUSION: The predictive score demonstrated great performance for identifying CSR of initially unresectable CRLM. Stratifying patients by the score, personalized treatment goals can be formulated before conversion therapy to improve clinical prognosis and reduce adverse events caused by ineffective treatment.

Intratumor APOL3 delineates a distinctive immunogenic ferroptosis subset with prognosis prediction in colorectal cancer.

With the essential role of lipid transporting signaling in cancer-related immunity, apolipoprotein L3 (APOL3), a member of the apolipoprotein L gene family, demonstrated significant modulation ability in immunity. However, the expression profile and critical role of APOL3 in colorectal cancer (CRC) remain unclear. This study aimed to investigate the prognostic significance of APOL3 expression and its biological predictive value in CRC. The study enrolled multiple cohorts, consisting of 911 tumor microarray specimens of CRC patients from Zhongshan Hospital, 412 transcriptional data from The Cancer Genome Atlas, and 30 single-cell RNA sequencing (scRNA-seq) from internal and external CRC patients. APOL3 mRNA expression was directly acquired from public datasets, and APOL3 protein expression was detected using immunohistochemistry. Finally, the associations of APOL3 expression with clinical outcomes, immune context, and genomic and ferroptotic features were analyzed. Low APOL3 expression predicted poor prognosis and inferior responsiveness to 5-fluorouracil-based adjuvant chemotherapy (ACT) and targeted therapy. APOL3 fosters an immune-active microenvironment characterized by the promotion of ferroptosis, downregulation of macrophages, and upregulation of CD8+ T cell infiltration. Moreover, the expression of APOL3 in CD8+ T cells is intrinsically linked to ferroptosis and immune activation in CRC. In summary, APOL3 serves as an independent prognosticator and predictive biomarker for immunogenic ferroptosis, ACT, and targeted therapy in CRC. Furthermore, the APOL3 signaling activator could be a novel agent alone or in combination with current therapeutic strategies for CRC.

Prognostic Value of Tumor Deposits in Stage III Colorectal Cancer Patients with Different N Stages: A Population-Based, Retrospective, Cohort Study.

PURPOSE: Tumor deposits (TDs) seem to be associated with the prognosis of patients with colorectal cancer (CRC). The goal of this study was to investigate the prognostic value of TDs among patients with stage III CRC at different N stages. METHODS: A retrospective analysis was performed on two independent cohorts of stage III CRC patients from the Surveillance, Epidemiology, and End Results (SEER) database (n = 8232) and the First Affiliated Hospital of Wenzhou Medical University (n = 423). Primary outcomes were overall survival (OS) and cancer-specific survival (CSS). RESULTS: Of 8232 patients in the SEER cohort, the presence of TDs revealed poorer 5-year OS rates and 5-year CSS rates in all N-stage subgroups. X-tile software identified 5 (5-year OS: P = 0.004; 5-year CSS: P < 0.001) as the optimal cutoff value for TD count in the TD-positive subgroup at the N2 stage. The OS (5-year OS: 62.0% vs. 42.0%, P < 0.001) and CSS (5-year CSS: 66.0% vs. 43.8%, P < 0.001) of patients with five or more TDs were significantly worse than those with one to four TDs in the N2 stage subgroups. Of 423 patients in the Wenzhou cohort, the 3-year OS rate for patients in the positive group was worse than that for patients in the negative group (88.7% vs. 94.3%, P = 0.015). CONCLUSIONS: TD count should be considered when evaluating the prognosis of patients with the N2 stage. Those with higher TD counts (≥ 5) might have a worse prognosis.

Inhibition of thioredoxin-1 enhances the toxicity of glycolysis inhibitor 2-deoxyglucose by downregulating SLC1A5 expression in colorectal cancer cells.

BACKGROUND: Targeting glycolysis in cancer is an attractive approach for therapeutic intervention. 2-Deoxyglucose (2DG) is a synthetic glucose analog that inhibits glycolysis. However, its efficacy is limited by the systemic toxicity at high doses. Understanding the mechanism of 2DG resistance is important for further use of this drug in cancer treatment. METHODS: The expression of thioredoxin-1 (Trx-1) in colorectal cancer (CRC) cells treated with 2DG was detected by Western blotting. The effect of Trx-1 on the cytotoxicity of 2DG in CRC cells was examined in vitro and in vivo. The molecular mechanism involved in Trx-1-mediated activation of the SLC1A5 gene promoter activity was elucidated using in vitro models. RESULTS: Inhibition glycolysis with 2DG increased the expression of Trx-1 in CRC cells. Overexpression of Trx-1 decreased the cytotoxicity of 2DG, whereas knockdown of Trx-1 by shRNA significantly increased the cytotoxicity of 2DG in CRC cells. The Trx-1 inhibitor PX-12 increased the cytotoxicity of 2DG on CRC cells both in vitro and in vivo. In addition, Trx-1 promoted SLC1A5 expression by increasing the promoter activity of the SLC1A5 gene by binding to SP1. We also found that the SLC1A5 expression was upregulated in CRC tissues, and inhibition of SLC1A5 significantly enhanced the inhibitory effect of 2DG on the growth of CRC cells in vitro and in vivo. Overexpression of SLC1A5 reduced the cytotoxicity of 2DG in combination with PX-12 treatment in CRC cells. CONCLUSION: Our results demonstrate a novel adaptive mechanism of glycolytic inhibition in which Trx-1 increases GSH levels by regulating SLC1A5 to rescue cytotoxicity induced by 2DG in CRC cells. Inhibition of glycolysis in combination with inhibition of Trx-1 or SLC1A5 may be a promising strategy for the treatment of CRC.

Deep radiomics-based fusion model for prediction of bevacizumab treatment response and outcome in patients with colorectal cancer liver metastases: a multicentre cohort study.

Background: Accurate tumour response prediction to targeted therapy allows for personalised conversion therapy for patients with unresectable colorectal cancer liver metastases (CRLM). In this study, we aimed to develop and validate a multi-modal deep learning model to predict the efficacy of bevacizumab in patients with initially unresectable CRLM using baseline PET/CT, clinical data, and colonoscopy biopsy specimens. Methods: In this multicentre cohort study, we retrospectively collected data of 307 patients with CRLM from the BECOME study (NCT01972490) (Zhongshan Hospital of Fudan University, Shanghai) and two independent Chinese cohorts (internal validation cohort from January 1, 2018 to December 31, 2018 at Zhongshan Hospital of Fudan University; external validation cohort from January 1, 2020 to December 31, 2020 at Zhongshan Hospital-Xiamen, Shanghai, and the First Hospital of Wenzhou Medical University, Wenzhou). The main inclusion criteria were that patients with CRLM had pre-treatment PET/CT images as well as colonoscopy specimens. After extracting PET/CT features with deep neural networks (DNN) and selecting related clinical factors using LASSO analysis, a random forest classifier was built as the Deep Radiomics Bevacizumab efficacy predicting model (DERBY). Furthermore, by combining histopathological biomarkers into DERBY, we established DERBY+. The performance of model was evaluated using area under the curve (AUC), sensitivity, specificity, positive predictive value, and negative predictive value. Findings: DERBY achieved promising performance in predicting bevacizumab sensitivity with an AUC of 0.77 and 95% confidence interval (CI) [0.67-0.87]. After combining histopathological features, we developed DERBY+, which had more robust accuracy for predicting tumour response in external validation cohort (AUC 0.83 and 95% CI [0.75-0.92], sensitivity 80.4%, specificity 76.8%). DERBY+ also had prognostic value: the responders had longer progression-free survival (median progression-free survival: 9.6 vs 6.3 months, p = 0.002) and overall survival (median overall survival: 27.6 vs 18.5 months, p = 0.010) than non-responders. Interpretation: This multi-modal deep radiomics model, using PET/CT, clinical data and histopathological data, was able to identify patients with bevacizumab-sensitive CRLM, providing a favourable approach for precise patient treatment. To further validate and explore the clinical impact of this work, future prospective studies with larger patient cohorts are warranted. Funding: The National Natural Science Foundation of China; Fujian Provincial Health Commission Project; Xiamen Science and Technology Agency Program; Clinical Research Plan of SHDC; Shanghai Science and Technology Committee Project; Clinical Research Plan of SHDC; Zhejiang Provincial Natural Science Foundation of China; and National Science Foundation of Xiamen.

Nuclear translocation of thioredoxin-1 promotes colorectal cancer development via modulation of the IL-6/STAT3 signaling axis through interaction with STAT3.

Background: Thioredoxin 1 (Trx-1) is a small redox protein predominantly localized in the cytoplasm. Its expression is increased in several cancers, including colorectal cancer (CRC). However, the function of Trx-1 translocation to the nucleus in cancer is not clear. In this study, we investigated the role of Trx-1 nuclear translocation in development of CRC. Methods: Expression of Trx-1 and STAT3 was analyzed by Western blot and immunofluorescence. Endogenous interaction of Trx-1, STAT3, and karyopherin α1 in CRC cells was analyzed by co-immunoprecipitation. Trx-1 and pSTAT3 nuclear staining in human CRC tissues was analyzed by immunohistochemistry. A mouse model of AOM/DSS induced colitis-associated cancer (CAC) was utilized to investigate the antitumor effect of PX-12, a Trx-1 inhibitor. A knockin mouse with the Txn1(KK81-82EE) mutation was generated via CRISPR/Cas9, and CAC was induced in knockin and wild-type mice. Results: Nuclear translocation of Trx-1 was induced by IL-6, and inhibition of this translocation reversed IL-6-induced epithelial-to-mesenchymal transition, invasion and metastasis. Karyopherin α1 was found to specifically mediate IL-6-induced translocation of the Trx-1-pSTAT3 complex into the nucleus. Nuclear Trx-1 expression was closely correlated with lymph node metastasis and distant metastasis in human CRC. In addition, nuclear staining of Trx-1 showed significant positive correlation with nuclear staining of pSTAT3 in human CRC tissues. PX-12, an inhibitor of Trx-1, significantly impaired the activation of STAT3 and suppressed the development of AOM/DSS-induced CAC in mice. Moreover, AOM/DSS-induced nuclear Trx-1 expression was suppressed in Txn1(KK81-82EE) mice, which inhibited STAT3 activation and cancer progression. Conclusions: These results provide new insights into the mechanisms of STAT3 activation triggered by IL-6 and identify nuclear translocation of Trx-1 as a potential therapeutic target for the treatment of CRC and CAC.

The Role of RNA Methylation Modification Related Genes in Prognosis and Immunotherapy of Colorectal Cancer.

Background: Researches showed RNA methylation genes can affect the prognosis of tumors. Thus, the study aimed to comprehensively analyze the effects of RNA methylation regulatory genes in prognosis and treatment of colorectal cancer (CRC). Methods: Prognostic signature associated with CRCs were constructed by differential expression analysis, Cox and Least Absolute Shrinkage and Selection Operator (LASSO) analyses. Receiver operating characteristic (ROC) and Kaplan-Meier survival analyses were used to validate the reliability of the developed model. Gene Ontology (GO), Gene set variation analysis (GSVA), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. Finally, normal and cancerous tissue were collected to validate gene by quantitative real-time PCR (qRT-PCR). Results: A prognostic risk model based on leucine rich pentatricopeptide repeat containing (LRPPRC) and ubiquitin-like with PHD and ring finger domains 2 (UHRF2) was constructed and relevant to the overall survival (OS) of CRC. Functional enrichment analysis revealed that collagen fibrous tissue, ion channel complex and other pathways were significantly enriched, which might help explain the underlying molecular mechanisms. There were significant differences in ImmuneScore, StromalScore, ESTIMATEScore between high- and low-risk groups (p < 0.05). Ultimately, qRT-PCR validation showed that a significant upregulation in the expression of LRPPRC and UHRF2 in cancerous tissue, which verified the effectiveness of our signature. Conclusion: In conclusion, 2 prognostic genes (LRPPRC and UHRF2) related to RNA methylation were identified by bioinformatics analysis, which might supply a new insight into the treatment and evaluation of CRC.

Identification of Senescence-Related Subtypes, the Development of a Prognosis Model, and Characterization of Immune Infiltration and Gut Microbiota in Colorectal Cancer.

Cellular senescence is associated with tumorigenesis, and the subtype and prognostic signatures of senescence-related genes (SRGs) in the tumor microenvironment (TME) and gut microbiota have not been fully determined. Analysis of 91 SRGs obtained from the GSEA and MSigDB, and mRNA sequencing of genes in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases enabled the identification of two distinct molecular types of colorectal cancer (CRC). Patient samples were clustered into two subtypes, with Kaplan-Meier survival analyses showing significant differences in patient survival between the two subtypes. Cluster C2 was associated with patient clinicopathological features, high immune score, high abundance of immune infiltrating cells and somewhat high abundance of bacteria. A risk model based on eight SRGs showed that a low risk score was characterized by inhibition of immune activity and was indicative of better prognosis in patients with CRC. In combination with clinical characteristics, risk score was found to be an independent prognostic predictor of survival in patients with CRC. In conclusion, the present study showed that senescence-related subtypes and a signature consisting of eight SRGs were associated with CRC patient prognosis, as well as with immune cell infiltration and gut microbiota. These findings may enable better prediction of CRC patient prognosis and facilitate individualized treatments.

A Novel Pyroptosis-Associated Gene Signature to Predict Prognosis in Patients with Colorectal Cancer.

Background: Pyroptosis is a form of cell death characterized by cell swelling and plasma membrane bubbling in association with inflammatory and immune responses. To date, the association between pyroptosis and colorectal cancer remains unclear. We aimed to establish a novel pyroptosis-associated model for the prognosis of colorectal cancer. Methods: Pyroptosis-related genes were extracted using Gene Set Enrichment Analysis. A least absolute shrinkage and selection operator regression model was constructed to identify a pyroptosis-related gene signature using the Cancer Genome Atlas and Gene Expression Omnibus databases. Then, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology and GSEA were performed to better understand the potential mechanisms and the functional pathways associated with pyroptosis involved in colorectal cancer. The relationship between the pyroptosis-related signature and immune infiltration was investigated using Cell-Type Identification by Estimating Relative Subsets of RNA Transcripts and MCPcounter. Results: A 12 pyroptosis-related gene signature was identified. Then, patients were classified into high- and low-risk groups. Kaplan-Meier and receiver operating characteristic analyses confirmed that the high-risk groups showed worse overall survival, progression-free survival, or relapse-free survival probability. Functional enrichment analysis showed that pyroptosis was associated with extracellular matrix-related pathways. Furthermore, the pyroptosis risk score was associated with immune infiltration. The low-risk group exhibited a higher percentage of plasma cells, CD4 T cells, activated dendritic cells, and activated mast cells. M2 macrophages and M0 macrophages were positively related to the risk score. Conclusion: Our research yielded a novel pyroptosis-related prognostic signature for colorectal cancer that was related to immune cell infiltration, and it provided an immunological perspective for developing personalized therapies.

Regorafenib in Refractory Metastatic Colorectal Cancer: A Multi-Center Retrospective Study.

Background: Regorafenib improves progression-free survival (PFS) and overall survival (OS) in patients with refractory metastatic colorectal cancer (mCRC). Here, we report the treatment patterns of regorafenib in the third- or late-line setting for mCRC in four centers in China. Patients and Methods: Patients with refractory mCRC in four centers in China administered regorafenib from February 1, 2018 to June 31, 2021 were enrolled. Patients were grouped into 3 cohorts, namely, the monotherapy (regorafenib alone), chemo (regorafenib plus chemotherapy), and immune [regorafenib plus anti-PD1 (programmed cell death 1) antibodies] groups. Demographic, clinical, survival and safety data were retrospectively analyzed. Results: A total of 177 patients were included in this study. Of them, 116 (65.5%) were treated with regorafenib alone, while 28 (15.9%) and 33 (18.6%) were administered regorafenib plus chemotherapy and anti-PD1 antibodies, respectively. The median followed-up time was 9.2 months. The disease control rate (DCR) was 40.7%. The median PFS (mPFS) was 2.43 months and the median OS (mOS) was 12.2 months. The immune group had longer median PFS (3.5 m vs. 2.2 m, p = 0.043) compared with the monotherapy group. Patients administered regorafenib plus chemotherapy had longer median OS (15.9 m vs. 8.4 m, p = 0.032) compared with the monotherapy group. Patients who began regorafenib treatment at 120 mg had longer median PFS and OS compared with those who began at 80 mg (PFS: 3.7 m vs. 2.0 m; p <0.001; OS: 13.4 m vs. 10.2 m; p = 0.005). Patients with a final dose of 120 mg had longer median PFS and OS compared with the 80 mg or less group (PFS: 5.0 m vs. 2.3 m; p = 0.045; OS: UR (unreach) vs. 10.9 m; p = 0.003). There were 87.0% (154/177) patients who experienced AEs. Three groups had similar rates of AEs (86.2% vs. 89.3% vs. 87.9%; p = 0.89). Conclusion: Patients administered regorafenib alone or regorafenib in combination with other agents were relieved to some extent, with a disease control rate of 40.7%. Regorafenib plus anti-PD1 antibodies showed better PFS, while regorafenib plus chemotherapy had the most benefit in OS. There was no significant difference among three groups in terms of AEs.

Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer.

Cetuximab is approved for the treatment of metastatic colorectal cancer (mCRC) with RAS wild-type. Nevertheless, the prognosis remains poor and the effectiveness of cetuximab is limited in KRAS mutant mCRC. Recently, emerging evidence has shown that ferroptosis, a newly discovered form of nonapoptotic cell death, is closely related to KRAS mutant cells. Here, we further investigated whether cetuximab-mediated regulation of p38/Nrf2/HO-1 promotes RSL3-induced ferroptosis and plays a pivotal role in overcoming drug resistance in KRAS mutant colorectal cancer (CRC). In our research, we used two KRAS mutant CRC cell lines, HCT116 and DLD-1, as models of intrinsic resistance to cetuximab. The viability of cells treated with the combination of RSL3 and cetuximab was assessed by the CCK-8 and colony formation assays. The effective of cetuximab to promote RSL3-induced ferroptosis was investigated by evaluating lipid reactive oxygen species accumulation and the expression of the malondialdehyde and the intracellular iron assay. Cetuximab therapy contributed to regulating the p38/Nrf2/HO-1 axis, as determined by western blotting and transfection with small interfering RNAs. Cetuximab promoted RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 in KRAS mutant CRC cells, and this was further demonstrated in a xenograft nude mouse model. Our work reveals that cetuximab enhances the cytotoxic effect of RSL3 on KRAS mutant CRC cells and that cetuximab enhances RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 axis through the activation of p38 MAPK.

PUM1 Is Overexpressed in Colon Cancer Cells With Acquired Resistance to Cetuximab.

BACKGROUND: Cetuximab is an effective antibody to treat colorectal cancer (CRC) by targeting the epidermal growth factor receptor (EGFR). However, the mechanisms of acquired resistance to cetuximab therapy, especially in patients without identifiable gene mutations, are not fully understood. METHODS: Our study investigated the role of pumilio RNA-binding family member 1 (PUM1) in cetuximab resistance. We established cetuximab-resistant colon cancer cell lines SW480R and Caco-2R and knocked out PUM1 and DEAD-box helicase 5 (DDX5) with the clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) system. To check cell proliferation, we used Cell Counting Kit-8. We performed qPCR and immunoblot to examine the levels of mRNAs and proteins for each cell line. RESULTS: Our data showed that PUM1 was upregulated in SW480R and Caco-2R cells with increased protein levels and cell proliferation, and PUM1 knockout reduced cell viability in the presence of cetuximab. We also found that PUM1 interacted with DDX5 in 3' untranslated region (UTR) and positively regulated its mRNA expression. Furthermore, suppression of DDX5 also decreased the proliferation of SW480R and Caco-2R cells. CONCLUSION: Our study suggests that PUM1 positively regulates DDX5 and acts as a promoter in cetuximab-resistant colon cancer cells.

Stress-induced phosphoprotein 1 facilitates breast cancer cell progression and indicates poor prognosis for breast cancer patients.

Breast cancer (BC) threatened the life health of a tremendous amount of the population, and the estimated number of death is still rising nowadays. We found that stress-induced phosphoprotein 1 (STIP1) is overexpressed in BC tissues compared to non-tumorous breast tissues. Our study is to validate the prognostic value of STIP1 and investigate its biological role in BC. We verified the upregulation of STIP1 in multiple databases, proved that STIP1 is upregulated in BC tissues and cell lines using real-time quantitative PCR (qRT-PCR). We used small interfering RNA to examine the function of STIP1 in BC cell lines (BT-549, MDA-MB-231, Hs-578 T) and explored the mechanism of function of STIP1 in BC cells using Western blotting and qRT-PCR. Analyses of multiple databases indicated that high STIP1 expression is a marker that effectively distinguishes BC patients from healthy control and predicts worse clinical outcomes in BC. The loss-of-function experiments showed that STIP1 silencing results in inhibition of cell proliferation and migration, inducing cell apoptosis, and S-phase arrest in vitro. Our study also showed that STIP1 downregulation inhibited the JAK2/STAT3 pathway and epithelial-mesenchymal transition process. Rescue experiments demonstrated that the oncogenic effect of STIP1 is partially dependent on mediating JAK2 expression. This study verified that STIP1 is an oncogenic gene that promotes BC progression and serves as a valuable diagnostic and outcome-related marker of BC.

Long Noncoding RNA PCAT18 Upregulates SPRR3 to Promote Colorectal Cancer Progression by Binding to miR-759.

BACKGROUND: Long noncoding RNAs (lncRNAs) play essential functions in the development of several cancers, including colorectal cancer (CRC). Nevertheless, how PCAT18 regulates CRC tumorigenesis remains unclear. In this research, we aimed to investigate the roles of PCAT18 in CRC. MATERIALS AND METHODS: qRT-PCR and Western blot were used to analyze RNA and protein levels. CCK8, colony formation, transwell and wound healing assays were utilized to analyze proliferation, migration and invasion. Luciferase reporter assay was used to analyze RNA interactions. RESULTS: PCAT18 was found to be highly expressed in CRC tissues and cells. PCAT18 level was positively correlated with lymph node metastasis and TNM stage. Functionally, PCAT18 silencing induced impairment of CRC proliferation, migration and invasion. Besides, PCAT18 was identified to inhibit miR-759. PCAT18 promotes SPRR3 expression through binding to miR-759. Furthermore, miR-759 inhibitors or SPRR3 ectopic expression partially rescued the abilities of proliferation, migration and invasion in CRC cells transfected with sh-PCAT18. CONCLUSION: Therefore, our study demonstrated that PCAT18 contributes to CRC progression through regulating miR-759/SPRR3 axis, which provides a new theoretical basis of explaining CRC tumorigenesis.

Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9.

Colorectal cancer (CRC) is a common malignant tumor in digestive tract with highly invasive and metastatic capacity. Drug sensitivity remains a significant obstacle to successful chemotherapy in CRC patients. The present study aimed to explore genes related to cetuximab (CTX) sensitivity in CRC by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Celigo image cytometer was used to detect suitable cells and optimal dosage of CTX. Inhibition rate of CTX on Caco-2 cells was evaluated by cell counting kit-8 (CCK-8) method before and after transfection. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was performed to explore suitable concentration of puromycin and multiplicity of infection (MOI). CRISPR-Cas9, sequencing data quality analysis and cell viability test were used for the selection of genes related to CTX sensitivity in CRC cells. Finally, the selected genes associated with CTX sensitivity in CRC cells were further validated by colony formation and CCK-8 assays. In the present study, Caco-2 cells had a better prolificacy, and CTX 100 µg/ml exhibited a good inhibition trend on the 7th and 14th days of infection. MTT assay indicated that the minimum lethal concentration of puromycin was 2.5 µg/ml. Forty-six candidate genes were preliminarily screened via sequencing data quality analysis. Subsequently, we found that knockout of any of the four genes (MMP15, MRPL48, CALN1 and HADHB) could enhance CTX sensitivity in Caco-2 cells, which was further confirmed by colony formation assay. In summary, MMP15, MRPL48, CALN1 and HADHB genes are related to the mediation of CTX sensitivity in CRC.

Autophagy regulates the therapeutic potential of adipose-derived stem cells in LPS-induced pulmonary microvascular barrier damage.

Adipose-derived stem cells (ADSCs) have been shown to be beneficial in some pulmonary diseases, and the paracrine effect is the major mechanism underlying ADSC-based therapy. Autophagy plays a crucial role in maintaining stem cell homeostasis and survival. However, the role of autophagy in mediating ADSC paracrine effects has not been thoroughly elucidated. We examined whether ADSCs participate in lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cell (PMVEC) barrier damage in a paracrine manner and illuminated the role of autophagy in regulating ADSC paracrine effects. PMVECs and ADSCs with or without autophagy inhibition were cocultured without intercellular contact, and the microvascular barrier function was assessed after LPS treatment. ADSC paracrine function was evaluated by detecting essential growth factors for endothelial cells. For in vivo experiments, ADSCs with or without autophagy inhibition were transplanted into LPS-induced lung-injury mice, and lung injury was assessed. ADSCs significantly alleviated LPS-induced microvascular barrier injury. In addition, ADSC paracrine levels of VEGF, FGF, and EGF were induced by LPS treatment, especially in the coculture condition. Inhibiting autophagy weakened the paracrine function and the protective effects of ADSCs on microvascular barrier injury. Moreover, ADSC transplantation alleviated LPS-induced lung injury, and inhibiting autophagy markedly weakened the therapeutic effect of ADSCs on lung injury. Together, these findings show that ADSC paracrine effects play a vital protective role in LPS-induced pulmonary microvascular barrier injury. Autophagy is a positive mediating factor in the paracrine process. These results are helpful for illuminating the role and mechanism of ADSC paracrine effects and developing effective therapies in acute lung injury.

Circular RNA hsa_circRNA_102958 promotes tumorigenesis of colorectal cancer via miR-585/CDC25B axis.

Purpose: Circular RNAs (circRNAs) are recently identified new noncoding RNAs and play an important role in tumorigenesis. Previous studies have indicated that hsa_circRNA_102958 is a potential diagnostic indicator in gastric cancer. However, its role in colorectal cancer (CRC) is poorly understood. Methods: qRT-PCR and ISH were used to test gene expression in tissues. Survival rate was analzyed by Kaplan-Meier curve. Luciferase reporter assay was used to determine the interaction between circRNA and miRNA or between miRNA and mRNA. Western blotting was used to test protein expression. CCK8 and colony formation assay was used to analyze proliferation. Transwell assay was used for migration and invasion determination. Results: In our research, we found that hsa_circRNA_102958 expression was significantly increased in CRC tissues, compared to adjacent normal controls. Increased hsa_circRNA_102958 levels in CRC patients indicated a poor prognosis. The effects of hsa_circRNA_102958 on CRC cell proliferation, migration and invasion were then determined by CCK8, colony formation and Transwell assays. We showed that hsa_circRNA_102958 silencing markedly suppressed CRC growth, migration and invasion. Furthermore, hsa_circRNA_102958 was identified as a sponge for miR-585. We demonstrated that hsa_circRNA_102958 promoted CDC25B expression through inhibiting miR-585 in CRC. Rescue assays illustrated that CDC25B overexpression reversed the suppressive effects of hsa_circRNA_102958 silencing on CRC. Conclusion: Taken together, our findings revealed the novel oncogenic roles of hsa_circRNA_102958 in CRC through miR-585/CDC25B axis.