Cervical cancer (CC) is the fourth most common and deadliest cancer worldwide [1]. The incidence and mortality of CC are significantly higher in China than in other countries, with distinct geographical characteristics, especially in Shanxi Province, which has the highest incidence of CC in China [2]. Cervical squamous cell carcinoma (SCC) is the most common type of CC, with approximately 80% of primary CC arising from pre-existing squamous cell dysplasia, and persistent human papillomavirus (HPV) infection is considered to the most important factor in the pathogenesis of SCC [3]. However, not all women infected with HPV can develop to SCC [1,4], because other triggers are required for the progression of SCC, such as smoking, folate deficiency (FD), ultraviolet radiation, immunosuppression, and pregnancy [5]. Therefore, exploring the synergistic factors and mechanisms of SCC progression may help us understand the causes of its progression and provide new measures for the early prevention and treatment of SCC.

Folic acid (vitamin 9) activates one-carbon metabolism and provides substrates for methylation reactions in cells, including DNA, RNA, histones, neurotransmitters, and other small molecule compounds [6,7] .FD may promote cancer progression by altering cellular environment and causing genomic instability, such as alterations in microRNAs (miRNAs) expression, genomic DNA methylation, and histone methylation [8], [9], [10]. Given the geographical characteristics of high SCC prevalence in Shanxi Province, we conducted a large-scale population-based study (40,000 people) in two high SCC prevalence areas (YangQu and JieXiu) in Shanxi Province and found that in addition to common risk factors, dietary folate and serum FD were closely associated with the progression of cervical intraepithelial neoplasia (CIN) [11], [12], [13]. Moreover, the most pathogenic HPV types in Shanxi Province are HPV 16, 58, 31, 33, and 52 [14], and maintaining normal folate levels reduces the risk of CIN and SCC in women infected with high-risk HPV(HR-HPV) [15]. These studies suggest that FD may serve as a synergistic factor in SCC caused by HR-HPV infection and that FD may be a geographical characteristic of SCC patients in Shanxi Province. Meanwhile, studies from other countries have also reported that folate levels are closely associated with CIN and SCC progression [16], [17], [18]. All these findings suggest that FD may be a major synergistic factor in the progression of SCC. However, these studies represent evidence that is confined to the epidemiological level, while the underlying molecular mechanisms of the relationship between FD and SCC remain unclear. miRNAs are short noncoding RNAs (containing approximately 22 nucleotides) that can regulate tumorigenesis by targeting key post-transcriptional genes in the DNA damage/repair pathways that in turn modulate genomic and chromosomal instability [19].Our previous study analyzed different grades of cervical lesions from FD and HPV16-infected patients in Shanxi Province [12,20] and found that miR-375 was the most significantly downregulated miRNA. miR-375 is a DNA methylation-regulated miRNA whose dysregulation may contribute to the pathophysiology of SCC [21]. Frizzled4 (FZD4) receptors is a member of the cancer driver G protein-coupled receptors (GPCRs), and activation of FZD4 and its downstream Wnt/β-catenin signaling pathway plays an important role in abnormal proliferation of cancer cells [22] . Although there have been some studies on miR-375 in different cancers [23], [24], [25], [26], [27], the regulatory mechanisms of folic acid and miR-375 in SCC remained unclear.

In the present study, we aimed to elucidate the mechanisms by which FD and miR-375 contribute to SCC progression. The results of this study may provide new insights into the role of folic acid in the prevention and treatment of SCC.