Abstract:

Objective To explore the prognostic value of long non-coding RNA （lncRNA） AL445524.1 in hepatocellular carcinoma （HCC） and its regulatory effect on liver cancer cells. Methods Based on the TCGA database， bioinformatics analysis was conducted to assess the expression levels of AL445524.1 in HCC. Using the median expression level of AL445524.1 as the cut-off value， 343 HCC patients were divided into high expression group and low expression group. Survival analysis was performed using Kaplan-Meier curves. Univariate and multivariate Cox proportional hazards regression models were employed to analyze the relationship between AL445524.1 expression， other clinical characteristics， and patients' prognosis， identifying independent risk factors for the prognosis of HCC patients. Based on the results of the multivariate analysis， patients with complete data were randomly divided into a testing set （n=215） and a training set （n=92） in a 7∶3 ratio using a random number table method， and a nomogram prognostic prediction model for HCC was constructed. Receiver operator characteristic （ROC） curves were plotted to calculate the area under the curve （AUC） to analyze and validate the predictive performance of the model. Functional enrichment analysis was conducted on the differentially expressed genes regulated by AL445524.1. The expression level of AL445524.1 in liver cancer cells was detected using RT-PCR. Human hepatoma cell lines HCCLM3 were divided into si-AL445524.1-1， si-AL445524.1-2， si-AL445524.1-3 and si-NC groups. The CCK-8 assay was used to assess cell proliferation capability； cell scratch and Transwell assays were performed to evaluate cell migration and invasion abilities； and flow cytometry was utilized to detect cell apoptosis. Results The expression of AL445524.1 in liver cancer tissues was significantly higher than that in para-carcinoma tissues （4.38±1.26 vs. 2.08±0.45， t=24.58， P<0.001）. Kaplan-Meier survival analysis showed that the 5-year overall survival （OS） rate for HCC patients in high AL445524.1 expression group （n=170） was 37.37%， and that of patients in low AL445524.1 expression group （n=173） was 58.38%， with a statistically significant difference （χ²=8.83， P=0.003）. Multivariate analysis showed that tumor recurrence （HR=2.58， 95%CI： 1.64-4.07， P<0.001）， clinical stage （HR=2.49， 95%CI： 1.63-3.81， P<0.001）， and AL445524.1 expression （HR=1.23， 95%CI： 1.06-1.41， P=0.010） were independent factors affecting the prognosis of HCC patients. A nomogram prognostic prediction model was constructed based on tumor recurrence， clinical stage， and AL445524.1 expression， with the model risk score calculated as： risk score=0.774×tumor recurrence+0.753×clinical stage+0.231×AL445524.1. The prediction model C-index values of 0.726， 0.660， and 0.678 in the training set， testing set， and overall set， respectively. ROC curve analysis showed that the AUC values for the 1-year OS rates in the training set， testing set， and overall set were 0.746， 0.630， and 0.684， respectively； the AUC values for the 3-year OS rates were 0.778， 0.736， and 0.743； and the AUC values for the 5-year OS rates were 0.794， 0.760， and 0.774. In the training set， the test set and the overall set， the predictive performance of the model score in predicting the 5-year OS rate of patients was superior to the individual predictions of AL445524.1 expression， clinical stage and tumor recurrence alone （all P<0.05）. The 5-year overall survival （OS） rates of high-risk group （n=154） and low-risk group （n=153） were 33.54% and 77.73%， respectively， with a statistically significant difference （χ²=28.97， P<0.001）. GO and KEGG enrichment analysis suggested that the differential expression of AL445524.1 genes in high and low expression groups was mainly related to lipid metabolism and oxidation. GSEA analysis showed that the oxidative phosphorylation pathway was significantly enriched in HCC patients with high expression of AL445524.1. In vitro experiments showed that AL445524.1 expression was higher in liver cancer cells （1.97±0.14） compared to normal liver cells （1.00±0.10）， with a statistically significant difference （t=11.62， P=0.007）. Silencing AL445524.1 could significantly inhibit the proliferation， migration， and invasion of liver cancer cells and promote apoptosis. In the CCK-8 proliferation experiment， the A₄₅₀ values of the si-NC， si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups after 24 hours were 0.433±0.012， 0.377±0.020， 0.383±0.020， and 0.423±0.005， respectively， with a statistically significant difference （F=20.51， P<0.001）. Additionally， the cell proliferation in the si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups was lower than that in the si-NC group （all P<0.001）. The cell scratch assay showed that the scratch healing rates of the above 4 groups were 33.60%±6.15%， 21.60%±4.30%， 26.40%± 4.60%， and 27.30%±2.60%， respectively， with a statistically significant difference （F=42.71， P<0.001）. The scratch healing rates of the si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups were also significantly lower than that of the si-NC group （all P<0.001）. Transwell migration and invasion experiments revealed that the number of migrated cells in the above 4 groups were 293.50±14.80， 110.50±10.28， 132.44±5.57， and 115.25±8.66， respectively， with a statistically significant difference （F=374.16， P<0.001）. The number of migrated cells in the si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups were significantly lower than that in the si-NC group （all P<0.001）. For the invasion assay， the number of invaded cells in the above 4 groups were 247.00±9.49， 119.00±5.57， 153.25±5.85， and 163.67±5.51， respectively， with a statistically significant difference （F=218.14， P<0.001）. The number of invaded cells in the si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups were also significantly lower than that in the si-NC group （all P<0.001）. Flow cytometry showed that the apoptosis rates of the above 4 groups were 1.70%±0.08%， 2.17%±0.11%， 2.38%±0.08%， and 2.02%±0.27%， respectively， with a statistically significant difference （F=29.36， P<0.001）. The apoptosis rate in the si-AL445524.1-1， si-AL445524.1-2， and si-AL445524.1-3 groups were significantly higher than that in the si-NC group （all P<0.001）. Conclusion AL445524.1 can serve as a prognostic marker for HCC. The AL445524.1-related prognostic prediction model demonstrates high accuracy in predicting the 5-year OS rate of HCC patients. Patients with high AL445524.1 expression have poorer survival prognosis. Silencing AL445524.1 can inhibit liver cancer cell proliferation， migration， and invasion while promoting apoptosis， which may be related to cellular lipid metabolism or oxidative phosphorylation.

Key words: Carcinoma， hepatocellular, lncRNA AL445524.1, Bioinformatics analysis, Cell prolifera-tion, Cell movement, Neoplasm invasiveness, Apoptosis