HNSCC development is a complicated process involving multi-factor participation, and miRNA plays a critical role in the regulation of HNSCC. Radiotherapy is a crucial method for the treatment of HNSCCs, including LSCC. Therefore, elucidating the radiotherapy mechanism in LSCC could reveal novel diagnostic and therapeutic approaches. The identification of new and effective radiation-related genes is also key to the successful treatment of LSCC. Thus, it is imperative to uncover novel molecules that will be beneficial in predicting the prognosis and radiotherapy response of laryngeal carcinomas. Ultimately, this will lead to more customized treatment and improved outcomes for patients with HNSCC .
The miR-125 family is related to the development and treatment of cancers and may even impact the survival outcomes of patients with NSCLC . Specifically, miR-125a-3p has been related to many tumors, including breast cancer, renal cancer, hepatocellular carcinoma, colorectal cancer and colon cancer [15–19]. Moreover, sponging miR-125a-3p in HNSCC may promote tumor growth through the regulation of FGFR1 expression . Another study indicated that miR-125b-1-3p had anticancer effects in lung cell carcinoma and was a diagnostic marker for pancreatic cancer [21, 22]. MiR-125b-2-3p is related to the prognosis of hepatocellular carcinoma and may predict chemotherapy outcomes in patients with colorectal cancer [23, 24]. Furthermore, miR-125b-2-3p inhibits the TNM-based progression of HNSCC . MiR-125b-5p plays an essential role in suppressing cancer progression and invasion in bladder cancer, LIHC, BRCA, and lung cancer, and is also related to drug resistance in colon cancer [26–29]. MiR-125b-5p may also promote the progression of LSCC and is an essential drug target [30, 31].
In this study, we first systematically studied the miR-125a family using the TCGA database and then performed pan-cancer analysis on all members of the family. MiR-125 expression differences were statistically significant in many cancers. All members of the miR-125 family exhibited significantly different expression levels in HNSCC. We then conducted a comprehensive and systematic analysis of the clinical parameters related to HNSCC, including the TNM stage, clinical stage, histologic grade, and effect of radiation therapy. MiR-125b-1-3p, miR-125b-2-3p, and miR-125b-5p were clinically significant for the diagnosis of T staging and M staging. Furthermore, all members of the miR-125 family, except miR-125a-5p, had diagnostic significance at the N1 and N2 stages but no diagnostic significance at the N3 stage. MiR-125b-1-3p, miR-125b-2-3p, and miR-125b-5p were significantly related to all cancer stages and tumor grades, except G4, in HNSCC. Furthermore, radiation therapy had a statistically significant effect on the expression of all members of the miR-125 family, except miR-125a-3p. Subsequently, we conducted a survival analysis of patients with LSCC and found that miR-125a-5p was related to the survival time of LSCC. Therefore, we further analyzed the correlation between miR-125a-5p and LSCC sensitivity to radiotherapy.
Several experiments have confirmed that miR-125a-5p influences tumor growth in cancers, such as COAD, LIHC, BRCA, PRAD, and cervical cancer [32–36]. In HNSCC, miR-125a-5p may inhibit growth, progression, and metastasis by targeting ERBB3 and promoting cell apoptosis . MiR-125a-5p was significantly differentially expressed between tumor tissue and normal tissue and was related to the occurrence, growth, and invasion of LSCC; thus, it may represent a new biomarker for biotherapy . Our research corroborates these conclusions and reveals the function of miR-125a-5p in laryngeal carcinoma for the first time. Our results demonstrated that the growth and invasion ability of hep-2 cells were decreased, and the radiosensitivity was increased through the overexpression of miR-125a-5p; therefore, miR-125a-5p may function as an antineoplastic gene in hep-2 cells. The GO terms confirmed that differentially expressed target genes were related to signal transduction by the p53 class mediator, resulting in cell cycle arrest and DNA damage response (DDR). In the KEGG analysis, apoptosis was significantly enriched. The results of the KEGG enrichment analysis were consistent with those of the GO enrichment analysis. Seven hub genes were obtained by Cytoscape. Among them, p53 is very highly correlated with human tumors. P53 expression not only promotes apoptosis, but also inhibits the growth of tumors. When cancer develops further, p53 gene mutations are inactivated and apoptosis is inhibited. After radiotherapy, p53-mediated apoptosis is initiated.
Radiotherapy can induce cell apoptosis and chromosomal damage. Typical biochemical changes during radiation-induced apoptosis include single-chain breaks, endonuclease activation, and DNA double-strand breaks (DSBs), which play an important role in radiotherapy . The radiosensitivity of tumor cells is closely related to the radiation effect. Micronucleus experiments demonstrated that the micronucleation rate in the Ad-miR-125a-5p group increased after radiotherapy, and the overexpressed miR-125a-5p promoted nuclear damage, resulting in apoptosis. Radiation therapy can lead to DNA double-strand damage in tumor cells, triggering DDR, initiating the cell's own DNA repair mechanism, and activating multiple related intracellular signal transduction pathways . During the repair of DNA DSBs, the DNA damage sensors H2AX, RNF8, RAD50, ATM, ATR, and XRCC4, as well as many other molecules, play important roles in the DDR process . Moreover, miRNA regulates the expression of target genes in the DDR pathway at the post-transcriptional level . The DNA repair mechanism of tumor cells is one of the main reasons for tumor radiation resistance. Indeed, some researchers have proposed that genes acting on DNA repair proteins, which thereby affect cellular DNA repair, may enhance the efficacy of radiation therapy for tumors .
Increasing evidence supports the hypothesis that miRNAs are involved in the regulation of DNA repair proteins, thereby affecting the sensitivity of tumor cells to radiotherapy. For example, miR-101 targets DNA-PKCs and ATM, making tumors more sensitive to radiation . Moreover, miR-210 and miR-373 are upregulated in hypoxic cells and regulate the DNA damage repair pathway . In this study, miR-125-5p reduced the repair ability of DNA damage by regulating the expression of rH2AX when DNA double-strand damage occurred. H2AX is a histone located in the nucleus of cells and is phosphorylated to form rH2AX when DNA strand damage is followed by DSB . rH2AX level decreases with DNA repair and is then dephosphorylated when repair is complete. rH2AX is therefore, an index of reactive DNA repair. In this study, we confirmed that, in cells overexpressing miR-125a-5p, rH2AX level was significantly increased, DNA repair was inhibited, DSBs were promoted, cell apoptosis was inhibited, and tumor growth was inhibited under X-ray radiation at 10 Gy, which enhanced tumor sensitivity to radiotherapy. In the course of tumor radiotherapy, p53 plays an important role in apoptosis. By activating or inhibiting a series of genes, wild-type p53 can promote tumor cell apoptosis and enhance the sensitivity of tumors to radiotherapy by inhibiting the tumor cell cycle and the repair of radiation damage in tumor cells . Thus, the wild-type p53 gene plays an important role in the control of cell cycle arrest, DNA repair, and apoptosis. However, after mutation, the p53 gene loses its normal function, and the apoptosis process of tumors is inhibited after radiotherapy, resulting in resistance to radiotherapy . Many studies have shown that wild-type p53 can increase the radiosensitivity of HNSCC, lung cancer, BRCA, and many other tumors .
A recent study found that the wild-type p53 gene induces apoptosis in 30% of tumor cells and reduces proliferation in 80% of tumor cells . Moreover, the experimental results showed that miR-125a-5p could improve the sensitivity of tumor cells to radiotherapy by upregulating p53 and enhancing apoptosis in lung cancer. Similarly, a previous study found that miR-125a-5p contributes to p53 activation . Following radiation therapy-induced DSB, two repair pathways are activated: homologous recombination and nonhomologous end joining (NHEJ); of these, the most important pathway is NHEJ, which catalyzes the random connection of the two ends of DSBs through DNA-PKCs, Ku70, Ku80, and XRCC4 to repair DNA . However, the p53 gene can inhibit the NHEJ repair system, so that the double-stranded broken DNA cannot be repaired and tumor cells can undergo apoptosis. Control of the cellular response to radiotherapy by inhibiting DDR has been a focus of current radiotherapy research. In this study, we found that apoptosis of hep-2 cells was significantly increased by radiation treatment after upregulation of miR-125a-5p, indicating that miR-125a-5p has a radiotherapy sensitization effect. P53 activation plays an important role in DNA damage transduction and a pivotal role in processing "sub-nodes" in the DNA damage signal network. Our study proved that miR-125a-5p can upregulate rH2AX and P53 and inhibit DNA damage repair. Under the condition of radiotherapy, DNA damage and miR-125a-5p can induce DSBs and rH2AX phosphorylation in tumors, as well as activate p53 through the DDR pathway, thereby inhibiting tumor cell repair and enhancing the radiotherapy effect of tumor cells.