STEM I

Abstract

Hepatocellular Carcinoma (HCC), a major subset of liver cancer, stands as a global health concern, accounting for over 700,000 annual deaths annually. Current treatments offer limited prognostic efficacy, leading to persistent complications and tumor recurrence. A deeper comprehension of novel targets against HCC progression is imperative due to ineffective clinical treatments. Currently, many cell transduction pathways have been identified and correlated to excess Collagen I production in HCC, as Collagen I is an indicator of HCC progression; however, the involvement of Transforming growth factor-β1 (TGF-β1), which plays an important role in HCC development, and the gene encoding Collagen type I (COL1A1) in HCC remains unclear. This study aims to elucidate the previously unknown relationship of the TGFβ pathway and the COL1A1 gene in HCC by testing how TGF-β1 regulates the expression level of COL1A1 and its impact on the proliferation and migration of hepatocellular carcinoma cells. HepG2 cells were cultured and COL1A1 was knocked down via siRNA transfection to assess changes in proliferation and migration rates of HCC, in vitro. These cells were then treated with TGF-β1. Based on the findings, COL1A1 is overexpressed in HCC. Upregulation of COL1A1 facilitates the proliferation and migration of HCC cells through the TGFβ pathway. The correlation of TGF-β1 and COL1A1 upregulation introduces a novel therapeutic target against HCC, deterring HCC cell progression by reducing ambient levels of collagen I in the carcinoma tissue.

The graphical abstract was created in BioRender (BioRender, n.d.).

Liver cancer is one of the most malignant types of cancer, accounting for over 700,000 annual deaths worldwide (Ferlay et al., 2015). The most common form of liver cancer is Hepatocellular Carcinoma (HCC), which has emerged as the third leading cause of cancer-associated mortality worldwide. Currently, strategies against HCC include transplantation and target drugs; however, these treatment options lack proper prognosis, causing complications and tumor recurrence to remain a common occurrence. The lack of effective clinical treatments requires a further understanding of novel targets against hepatocellular carcinoma progression. Extracellular Matrix The extracellular matrix (ECM), a three dimensional structure with distinct biochemical and biomechanical properties, is an important component of the tumor microenvironment. Changes in the composition of the ECM have been shown to promote tumor growth in HCC (Khalfallah et al., 2011). Collagens are essential in ECM and can regulate tumor cell behavior (Wang et al., 2020). Collagen Type I is a major component of the liver ECM and is significantly upregulated in 83.7% of human liver cancer specimens compared with their adjacent non tumor tissue (Liu et al., 2021). Excess collagen in the tumor microenvironment creates a cycle of upregulation, further elevating levels of collagen I in the HCC microenvironment. The type I collagen network helps the basement membranes interact with nearby cells, playing a crucial role in cell migration and proliferation (Wang et al., 2020). The COL1A1 gene, alongside another alpha1 and alpha2 chain, provides instructions for making one component of type I collagen, which is upregulated in HCC as HCC progresses towards late stage carcinoma (Alomowitch et al., 2009). The upregulation of COL1A1 can generate a modified extracellular matrix environment that promotes the survival, proliferation, metastasis, and invasion of liver cancer cells (Wang et al., 2020). Therefore, the knockdown of the COL1A1 gene is a promising target to reduce HCC cell progression and moderate disease progression. TGF-β1 The process of HCC initiation, development, and progression involves various complex signaling pathways and networks, which are controlled by several oncogenes and tumor suppressor genes. Included in these pathways is the Transforming growth factor-β (TGF-β) signaling pathway, which plays important roles in several cellular processes, including proliferation and migration (Gonzalez-Sanchez et al., 2021). The TGF-β pathway’s involvement in cancer progression presents as an excellent therapeutic target. In the TGF-β pathway, Transforming growth factor-β1 (TGF-β1) binds to the TGF-β1 receptor, which induces the phosphorylation of Smad2/3 proteins. Phosphorylated Smad2/3 form a complex with Smad4, which translocates to the nucleus and regulates gene transcription through the interaction with various transcription factors (Ezzoukhry et al., 2016). TGF-β1 and Smad proteins are highly expressed in HCC cell lines and tissues, and play a significant role in the transcription of several genes, including COL1A1 (Shi et al., 2020). It has been identified that TGF-β1 stimulates the expression of COL1A1 and COL1A2 genes at the transcriptional and protein level; however little is known about TGF-β1’s relationship with COL1A1 and collagen I upregulation with respect to specific regulatory mechanisms relevant to HCC (Cutroneo et al., 2007). This study focuses on the role TGF-β1 plays in COL1A1 upregulation in HCC via the SMAD pathway.

Equipment and Materials
All materials required for this project were provided and paid for by Dr. Mullen’s Research Laboratory in the Department of Medicine/Division of Gastroenterology at UMass Chan Medical School. HepG2 cells were purchased from the American Type Culture Collection (ATCC) (HEPG2 - HB-8065 | ATCC, n.d.). The HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with GlutaMAX™, 10% Fetal Bovine Serum (FBS), and 1% Penicillin-Streptomycin, purchased from ThermoFisher. 1X Phosphate Buffered Saline (PBS), Dulbecco's Phosphate Buffered Saline (DPBS), and 0.25% Trypsin-EDTA, phenol red were purchased from ThermoFisher. Opti-MEM™ was also purchased from ThermoFisher. Dharmafect-1 was purchased from Fisher Scientific. SiRNA smart pool against human COL1A1 and non-targeting control #5 were purchased from Dharmacon (Horizon Discovery Group plc, Level Biotechnology, Inc, New Taipei City, Taiwan). MTT Cell Viability Assay was purchased from Thermo Fisher, and data was collected using a plate absorbance reader. Human TGF-beta 1 Recombinant Protein and COL1A1 antibody were purchased from Thermo Fisher. A nanometer, cell counter, and imaging software was also utilized to collect results.

In Silico Analysis of Cancer Expression
COL1A1 and TGF-β1 gene expression profiling and correlative studies were performed using the Gene Expression Omnibus (GEO) human hepatocellular carcinoma microarray dataset and The Cancer Genome Atlas (TCGA) liver cancer hepatocellular carcinoma (LIHC) cohort. Data from TCGA was analyzed using the TCGA portal using the University of California Santa Cruz (UCSC) Xena functional genomics explorer (Ma et al., 2019).

Cell Culture
The HepG2 cells were cultured at 37°C, in a 5% humidified carbon dioxide incubator. Cells were cultured in media composed of DMEM, 10% FBS, and 1% penicillin-streptomycin, and were grown for 48 hours in a 10 cm culture dish. Media was changed daily, and cells were split weekly in accordance with assays.

Testing COL1A1 siRNA knockdown Efficiency
Prior to conducting the proliferation and migration assays, the knockdown efficiency of COL1A1 siRNA was confirmed. Reverse siRNA transfection was utilized by plating COL1A1 siRNA and Non-targeting control (NTC) siRNA in 12-well plates. NTC and COL1A1 siRNA were mixed with 6 μL of Dharmafect-1 and 1188 μL of optimem. 478.8 μL of siRNA/transfection solution composed of 50 μM NTC5 siRNA, DPBS, and Optimem was added to each well of a 12-well dish. HepG2 cells were transferred and resuspended in 8640 μL of 16% FBS. 720 μL of HepG2 cells were added to each well, and cells were incubated in siRNA/transfection solution for 72 hours at 37°C before harvesting. After treatment, cells were harvested, and total RNA was extracted using TRIzol reagent. After following harvesting protocols, mRNA levels of COL1A1 were measured using a quantitative real-time polymerase chain reaction (qRT-PCR).

Cell Viability Assay
The MTT solution was prepared by mixing MTT with culture media. HepG2 cells were seeded in a medium containing 10% FBS 24 hours before performance of assay. 50 µL of serum-free media and 50 µL of MTT solution was added into each well. The plate was incubated at 37°C for 3 hours, and after incubation 150 µL of MTT solvent was added into each well. Absorption was read at OD=540 within 1 hour of added solvent. Cell proliferation analysis was conducted by averaging the duplicate reading for each sample and subtracting the culture medium background from the assay reading to get the correct absorbance proportional to cell number. siRNA knockdown efficacy was quantified using qtPCR.

Wound Healing Migration Assay
Cells were seeded in 6-well plates with medium containing 10% FBS, then cultured to 95–100% confluence. A scratch along the center of the confluent adherent cells layer was made with a sterile p10 pipette tip. Cell migration images were captured at 0, 12, 24 and 48 hours after the scratch was made under a microscope and analyzed using microscopy imaging analysis.

Western Blotting Analysis
Cellular protein lysates were isolated using a Protein Extraction Kit. An equal amount of protein lysate sample was loaded in each lane and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) along with a molecular weight marker following manufacturers instructions. Relative band intensity was analyzed using a Bio-Rad image software.

Statistical Tests
All assays were performed in triplicate. Comparisons between control and target groups with normal distribution between the groups was tested using a t-test or one-way ANOVA test. Comparison of means without a normal distribution between the groups was done using Mann-Whitney U-test or Kruskal-Wallis nonparametric tests. A p-value of < 0.05 was considered statistically significant.