Identification and quantification of serum KIN17 protein based on ELISA assay and exploring its clinical diagnostic value in liver cancer.

It has been well-elaborated that KIN17 protein is closely related to the expression, development and prognosis of liver cancer; however, till date, there has been no study about detecting the KIN17 protein in serum, which is important to developing clinical applications. The objective of this work is to detect serum KIN17 protein by the ELISA method and to explore the diagnostic significance of the KIN17 protein in liver cancer. First, we verified the ELISA method for serum KIN17 measurement according to five aspects: accuracy, precision, specificity, stability and detection limit. Results illustrate that the recovery rate of the ELISA method can be controlled between 90% and 110%, the variation coefficient of intra-assay can be controlled within 16%, and the variation coefficient of inter-assay can be controlled within 10%. There is no non-specific reaction with common tumor markers, and the detection limit can reach 0.125 ng mL-1. The results show that the KIN17 protein can be detected by ELISA, and there is a significant rise in KIN17 concentration in a liver cancer group compared with a healthy group, whose average concentrations are 1.730 ng mL-1 and 0.3897 ng mL-1, respectively. On this basis, we hypothesize that the serum KIN17 protein can serve as a potential biomarker of liver cancer and be measurable with the verified ELISA system after specific ultrafiltration and centrifugation, which is of great significance for the diagnosis and treatment of liver cancer.

KIN17 functions in DNA damage repair and chemosensitivity by modulating RAD51 in hepatocellular carcinoma.

The limited response of hepatocellular carcinoma (HCC) to chemotherapy drugs has always been a bottleneck in therapy. DNA damage repair is a major reason for chemoresistance. Previous studies have confirmed that KIN17 affects chemosensitivity. In this study, we examined the impact of KIN17 on chemotherapy response and DNA repair in HCC cells treated with oxaliplatin (L-OHP). We evaluated the expression and biological roles of KIN17 in HCC using bioinformatic analysis. The correlation between KIN17 and RAD51, particularly their nuclear expression levels, was evaluated using immunofluorescence, immunoblotting after nucleocytoplasmic separation in HCC cells, and immunohistochemistry of mouse xenograft tumors and human HCC tissues. The results indicated a significant increase in KIN17 expression in HCC tissues compared to normal tissues. The GSEA analysis revealed that upregulation of KIN17 was significantly associated with DNA damage repair. Knockdown of KIN17 led to increased DNA damage and reduced cellular survival after exposure to L-OHP. On the other hand, overexpression of KIN17 was linked to decreased DNA damage and improved cell survival following L-OHP treatment. Further experiments indicated that KIN17 affects the expression of RAD51, particularly in the nucleus. KIN17 plays a crucial role in influencing the sensitivity of HCC to chemotherapy by triggering the DNA repair response. Increased expression of KIN17 is associated with a poor prognosis for HCC patients, indicating that KIN17 could serve as a prognostic marker and therapeutic target for HCC.

Extraction-free, immuno-RPA-CRISPR/Cas13a-based one-pot detection of glypican-3 directly from extracellular vesicles.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI). It is not found in healthy adult liver but is overexpressed in human hepatocellular carcinoma (HCC). The protein marker GPC3 on extracellular vesicles (GPC3+ EVs) is also useful for HCC detection. Nevertheless, the absence of practical and dependable quantitative techniques to evaluate EVs proteins prevents their clinical implementation. RESULTS: Here, using an immuno-recombinase polymerase amplification (immuno-RPA) process and dual amplification of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we firstly create an extraction-free one-pot immuno-RPA-CRISPR (opiCRISPR) for the direct and extremely sensitive detection of EVs proteins. The EVs protein-targeted detection probe is amplified by RPA to generate a long repetitive sequence containing multiple CRISPR RNA (crRNA) targeting barcodes, and the signal is further amplified by the CRISPR-Cas13a side-chain cleavage activity to generate a fluorescent signal. The results show that circulating extracellular vesicle GPC3 (eGPC3) levels are a reliable marker for GPC3 expression in tumor, opening up new avenues for tumor diagnosis. SIGNIFICANCE AND NOVELTY: We created an eGPC3 assay based on the CRISPR-Cas13a system, and successfully study the significance of extracellular vesicle GPC3 markers in hepatocellular carcinoma.

Current methods for the detection of glypican-3.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI), widely expressed in human embryos, and is undetectable in healthy adult liver but overexpressed in human hepatocellular carcinoma (HCC). Therefore, accurate and sensitive detection of GPC3 is critical for disease diagnosis. In recent years, a series of methods have been developed for the highly sensitive detection of GPC3, but there is a lack of reviews on recent advances in GPC3-related assays. In this review, we provide the recent advances in GPC3 detection and GPC3 concentration detection, mainly in terms of various optical sensor-based assays and electrochemical assays, and also provide new insights into the challenges and future directions of the field.

Hypoxia-driven ncRNAs in breast cancer.

Low oxygen tension, or hypoxia is the driving force behind tumor aggressiveness, leading to therapy resistance, metastasis, and stemness in solid cancers including breast cancer, which now stands as the leading cause of cancer-related mortality in women. With the great advancements in exploring the regulatory roles of the non-coding genome in recent years, the wide spectrum of hypoxia-responsive genome is not limited to just protein-coding genes but also includes multiple types of non-coding RNAs, such as micro RNAs, long non-coding RNAs, and circular RNAs. Over the years, these hypoxia-responsive non-coding molecules have been greatly implicated in breast cancer. Hypoxia drives the expression of these non-coding RNAs as upstream modulators and downstream effectors of hypoxia inducible factor signaling in the favor of breast cancer through a myriad of molecular mechanisms. These non-coding RNAs then contribute in orchestrating aggressive hypoxic tumor environment and regulate cancer associated cellular processes such as proliferation, evasion of apoptotic death, extracellular matrix remodeling, angiogenesis, migration, invasion, epithelial-to-mesenchymal transition, metastasis, therapy resistance, stemness, and evasion of the immune system in breast cancer. In addition, the interplay between hypoxia-driven non-coding RNAs as well as feedback and feedforward loops between these ncRNAs and HIFs further contribute to breast cancer progression. Although the current clinical implications of hypoxia-driven non-coding RNAs are limited to prognostics and diagnostics in breast cancer, extensive explorations have established some of these hypoxia-driven non-coding RNAs as promising targets to treat aggressive breast cancers, and future scientific endeavors hold great promise in targeting hypoxia-driven ncRNAs at clinics to treat breast cancer and limit global cancer burden.

KIN17 promotes cell migration and invasion through stimulating the TGF-ß/Smad2 pathway in hepatocellular carcinoma.

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-ß/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-ß/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-ß/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Fe-catalyzed Decarbonylative Alkylative Spirocyclization of N-Arylcinnamamides: Access to Alkylated 1-Azaspirocyclohexadienones.

For the convenient introduction of simple linear/branched alkyl groups into biologically important azaspirocyclohexadienones, a practical Fe-catalyzed decarbonylative cascade spiro-cyclization of N-aryl cinnamamides with aliphatic aldehydes to provide alkylated 1-azaspiro-cyclohexadienones was developed. Aliphatic aldehydes were oxidative decarbonylated into primary, secondary and tertiary alkyl radicals conveniently and allows for the subsequent cascade construction of dual C(sp3)-C(sp3) and C=O bonds via radical addition, spirocyclization and oxidation sequence.