Unpacking Sorafenib Resistance in HCC: A Cheeky Look With a Side of Science
Ah, hepatocellular carcinoma (HCC), the life of the party when it comes to cancer! Ranked fourth in causing worldwide fatalities, it’s like a grim reaper wearing a lab coat, only adding to humanity’s woes. Enter sorafenib, the celebrated tyrosine kinase inhibitor (TKI) capable of taking on advanced HCC. Unfortunately, feeling like a celebrity doesn’t mean everyone can pull off the look, as it turns out that only 35–43% of HCC patients respond to our star drug before relapsing like a 90s pop sensation looking for a comeback. So, what gives?
Understanding Sorafenib Resistance: The Plot Thickens
Sure, those pesky clinical symptoms of HCC can be as sneaky as a cat burglar. By the time the diagnosis rolls in, it’s like being handed the wrong ticket to a concert — only to find that all the seats are sold out. In comes the dilemma of drug resistance; here it stands tall, blocking Sorafenib like a bouncer at an exclusive club. Ironically, after a rather disappointing 2.8-month survival advantage, most patients kick and scream at the mere mention of sorafenib. Time to reveal the culprits behind this resistance!
Ferroptosis: The New Buzzword in Cell Death
Let’s sprinkle some science magic on it, shall we? Enter ferroptosis, the new kid on the cell death block. Thanks to iron overload and the production of reactive oxygen species (ROS), it orchestrates a form of controlled cellular self-destruction. Who knew iron could be so dramatic? But hold your horses; as it turns out, key players like ferritin heavy chain 1 (FTH1), DMT1, and NQO1 are involved, too, playing a game of “Who’s Who?” while subtly nudging cells toward resistance. NQO1, the grumpy old contrarian, actively scours for lipid peroxides, acting like a stubborn toe-stubber in the fancy, inevitability dance of ferroptosis!
Non-Coding RNAs Take Center Stage
Now, let’s chat about the ever-mysterious non-coding RNAs (ncRNAs). Think of them as the quirky side characters who often steal the spotlight in a movie. Enter circular RNAs (circRNAs) and microRNAs (miRNAs), who clearly have no plans of playing the background. The plot twist here? These little segments have been implicated in the drama that is cancer progression, all while forming complex networks like a good old-fashioned soap opera.
The ceRNA Theory: A Salty Love Triangle
Under the ceRNA theory, circRNAs play the part of the “sponge,” soaking up miRNAs’ potential destructive plans like a full paper towel during a kitchen disaster. The interactions are mesmerizing! You have circRNAs happily tying the knot with miRNAs, swinging the balance in favor of their mRNA targets—like a clumsy matchmaker awkwardly shaking hands between parties only to end up creating a scenario where everyone’s getting tangled up.
The Heavyweights of HCC Resistance
Recently, innovative research uncovered the heavyweights in the sorafenib resistance arena: circ_0001944 and its downregulated pals circ_0078607 along with miR-1292-5p. Confessions of a romance gone wrong, if you will! This merry band of ribonucleic acid molecules engages in a lovely fight, with circ_0001944 hogging the spotlight by antagonizing miR-1292-5p and consequently influencing the enigmatic FBLN2 gene.
What’s FBLN2, you ask? Oh, just the landlord in charge of extracellular matrix homes–keeping things orderly while helping cells adhere. Advertising itself as a tumor suppressor in various cancers, while somehow managing to avoid HCC like it’s allergic to drama. What a character indeed!
A Tangled Web of Interactions
In this fascinating play of interactions, circ_0001944 mitigates ferroptosis, diplomatically steering clear of its bustling role. The role of the miR-1292-5p/FBLN2 axis cannot be cast away like a bad audition tape. As this axis in the HCC drama thickens, we uncover how this molecular soap opera serves as an astonishing revelation of how the suppression of ferroptosis fuels sorafenib resistance.
The Bottom Line: Moving From Theory to Therapy
What’s the takeaway from all this science-laden mischief? It seems that circ_0001944’s nefarious influence on ferroptosis through the miR-1292-5p/FBLN2 axis isn’t just a dramatic twist but could be the key to untangling sorafenib resistance. So, the next time someone mentions circular RNAs, raise a toast to their complexity and the fact that they still seem to be the ones calling the shots in this heavy-hitting oncogenic tale!
Remember, like every great story, this one requires clinical validation! Keep your eyes peeled for the evolution of this narrative. Who knows, the ending might just surprise us!
Introduction
Hepatocellular carcinoma (HCC) remains one of the leading health challenges, ranking as the fourth principal cause of mortality globally. Most HCC patients are often diagnosed at advanced stages, where clinical symptoms are typically subtle or undetectable. Among the treatment options, Sorafenib, a tyrosine kinase inhibitor (TKI), has served as the cornerstone drug to combat advanced HCC. However, many patients receiving this treatment express dissatisfaction with clinical outcomes as only approximately 35–43% of HCC individuals show a positive response to Sorafenib, and a significant proportion experience a relapse within just six months post-treatment. Furthermore, sorafenib-resistant patients only gain a meager survival advantage of around 2.8 months after treatment. Hence, overcoming drug resistance presents a critical challenge to effective sorafenib-based interventions in HCC, necessitating urgent research to uncover the mechanisms behind sorafenib resistance and to identify strategies to enhance drug effectiveness.
Ferroptosis, a regulated form of cell death characterized by iron accumulation and elevated reactive oxygen species (ROS) production, has recently garnered attention. Marked by the depletion of Ferritin Heavy Chain 1 (FTH1) and concomitant increases in Divalent metal-ion transporter-1 (DMT1) and Transferrin Receptor 1 (TFR1), ferroptosis leads to iron overload and lipid peroxidation. Additionally, Quinone oxidoreductase 1 (NQO1) serves as a protective scavenger, neutralizing lipid peroxides and ROS, hence antagonizing the ferroptosis process. Recent studies have unveiled that inhibition of ferroptosis correlates with drug resistance in various cancer types, including cisplatin resistance in gastric cancer and gemcitabine resistance in pancreatic cancer.
In the realm of cancer research, non-coding RNAs (ncRNAs), particularly circular RNAs (circRNAs) and microRNAs (miRNAs), are playing an increasingly pivotal role in regulating tumorigenesis both at transcriptional and post-transcriptional levels. CircRNAs, distinguished by their unique covalently closed circular structure formed during pre-mRNA splicing, exhibit remarkable stability against degradation. Similarly, miRNAs are critical regulators of gene expression in tumor cells. Utilizing the competitive endogenous RNA (CeRNA) hypothesis, circRNAs can act as molecular sponges for miRNAs, thereby modulating the expression and activity of target genes.
The involvement of circRNAs in various cancers has been well-documented, illustrating both oncogenic and tumor-suppressive properties depending on the context. For instance, Circ-E-Cad demonstrates oncogenic functions in glioblastoma, while Circ-Foxo3 has been associated with promoting apoptosis in breast cancer through interactions with key proteins including MDM2 and p53. Furthermore, the modulation of specific circRNA dynamics has been linked to metabolic reprogramming, immune microenvironment alterations, tumor progression, and metastasis in HCC. Yet, the precise mechanisms by which circRNAs contribute to sorafenib resistance through the modulation of ferroptosis in HCC remain poorly understood.
This study identified 70 upregulated and 45 downregulated circRNAs in SMMC7721/SOR cells, alongside 20 upregulated and 13 downregulated circRNAs in Huh7/SOR cells through circRNA Sequencing (circRNA-Seq). Correspondingly, 91 upregulated and 90 downregulated miRNAs in SMMC7721/SOR cells, as well as 50 upregulated and 38 downregulated miRNA clusters in Huh7/SOR cells, were determined via miRNA Sequencing (miRNA-Seq). Notably, circ_0001944 and circ_0078607 exhibited increased expression in sorafenib-resistant HCC cells, contrasting with downregulated expressions of circ_0002874 and circ_0069981. Similarly, a collection of eleven upregulated miRNAs (including miR-193a-5p, miR-197-3p, and others) and three downregulated miRNAs (miR-1292-5p being one of the key indicators) were identified.
Among these differentially expressed non-coding RNAs, circ_0001944 has been conclusively determined to target miR-1292-5p using comprehensive predictive models, followed by experimental validation like RNA binding protein immunoprecipitation (RIP) and dual luciferase reporter assays. Notably, findings confirm the regulatory interplay exists where circRNA and miRNA synergistically manage mRNA expressions following the ceRNA theory framework. The investigation culminated in a robust network analysis of “circ_0001944-miR-1292-5p-mRNA,” with particular interest surrounding target genes such as FBLN2, HRH3, GSTP1, and others which reside in close proximity to the established regulatory axis.
A crucial part of this analysis revealed that miR-1292-5p directly targets the 3’-noncoding region of FBLN2 to modulate its expression. Additionally, multiple functional assays, including mRNA-Seq and pathway enrichment analyses, established that FBLN2 influences ferroptosis pathways via the upstream regulatory effects exerted by miR-1292-5p. Thus, the trajectory of circ_0001944 was effectively mapped, illuminating its role in inhibiting ferroptosis and contributing to the sorafenib resistance narrative in HCC.
Materials and Methods
Cell lines utilized in this study included HCC lines SMMC7721 and Huh7 acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China). Sorafenib-resistant variants were established through a methodical induction protocol. Sorafenib and Ferrostatin-1 were obtained from Sigma-Aldrich. Various plasmids, such as Len-circ_0001944-shRNA and miR-1292-5p mimics, were sourced from GenePharma Co., Ltd (Shanghai, China), while Lipofectamine™3000 facilitated vector transfections into HCC cells.
Prior to sequencing, comprehensive RNA quantification processes were employed, involving checks for contamination and degradation via 1% agarose gel electrophoresis. RNA purity and concentration confirmation followed using NanoPhotometer® spectrophotometers. Ribosomal RNA (rRNA) removal was performed utilizing the Epicentre Ribo Zero rRNA Removal Kit, and library preparation ensued with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina.
For raw data analysis, clean reads were generated and evaluated using proprietary scripts, ensuring high-quality data for downstream analysis. Various expression analyses were performed using the DESeq2 R package, setting a threshold for significant differential expression.
The identification of target genes was conducted to elucidate the intricate relationships among differentially expressed circRNAs, miRNAs, and corresponding mRNAs through overlapping analyses. This culminated in the establishment of the “circRNA-miRNA-mRNA” regulatory network, depicting how these molecules interact in promoting sorafenib resistance. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses further enriched understanding of the biological pathways involved.
Results
RNA sequencing for both sensitive and resistant HCC cells revealed vital information regarding the role of non-coding RNAs in promoting drug resistance. The determination of differentially expressed ncRNAs (DENs offers a foundation for targeting these molecular players in therapeutic contexts.
To further substantiate the role of circ_0001944 and miR-1292-5p, experiments demonstrated that circ_0001944 could significantly enhance sorafenib resistance through inhibition of ferroptosis, highlighting its potential as a therapeutic target in HCC. Insights drawn from this study enhance the understanding of drug resistance mechanisms, particularly emphasizing the regulatory networks involving non-coding RNAs and their role in HCC progression and therapeutic response.
