Novel Insights into miR-944 in Cancer

Simple Summary miR-944 is localized in intron 4 of TP63. ΔNp63 in intron 3 of TP63 recruits the transcription factor AP-2 to promote miR-944 gene expression, which mediates epidermal differentiation induction by ΔNp63. miR-944 is dysregulated in various cancers. In squamous cell carcinoma. miR-944 can target and inhibit 27 protein-coding genes, thereby regulating cell cycle, proliferation, apoptosis, epithelial mesenchymal transition, cancer cell invasion and migration, and other cell behaviors. The genes targeted by miR-944 are involved in three signaling pathways, including the Wnt/β-catenin pathway, Jak/STAT3 pathway, and PI3K/AKT pathway. miR-944 was regulated by a total of 11 competing endogenous RNAs, including 6 circular RNAs and 5 long non-coding RNAs. Abnormally expressed miR-944 can act as an independent prognostic factor and is closely related to tumor invasion, lymph node metastasis, TNM staging, and drug resistance. miR-944 is expected to become a critical biomarker with great clinical application value in cancer. Abstract miRNA is a class of endogenous short-chain non-coding RNAs consisting of about 22 nucleotides. miR-944 is located in the fourth intron of the TP63 gene in the 3q28 region. miR-944 is abnormally expressed in cancers in multiple systems including neural, endocrine, respiratory, reproductive, and digestive systems. miR-944 can target at least 27 protein-coding genes. miR-944 can regulate a series of cell behaviors, such as cell cycle, proliferation, invasion and migration, EMT, apoptosis, etc. miR-944 participates in the networks of 11 ceRNAs, including six circRNAs and five lncRNAs. miR-944 is involved in three signaling pathways. The abnormal expression of miR-944 is closely related to the clinicopathological conditions of various cancer patients. Deregulated expression of miR-944 is significantly associated with clinicopathology and prognosis in cancer patients. In addition, miR-944 is also associated with the development of DDP, RAPA, DOX, and PTX resistance in cancer cells. miR-944 is involved in the anticancer molecular mechanisms of matrine and Rhenium-liposome drugs. In conclusion, this work systematically summarizes the related findings of miR-944, which will provide potential hints for follow-up research on miR-944.


Introduction
microRNAs (miRNAs) are endogenous short non-coding RNAs of approximately 22 nt that typically target the 3' untranslated region (3'-UTR) of mRNAs [1], thereby inhibiting the function of protein-coding genes [2]. Dysregulation of miRNAs is often associated with the malignant transformation of cells, thereby participating in biological processes that promote cancer progression, metastasis, and treatment resistance [3,4]. miR-944 is located in the fourth intron of tumor protein p63 (TP63) in the chromosome 3q28 region [5] and produced at the 3' end of the stem-loop structure of pre-mir-944. miR-944 is aberrantly expressed in more than 10 cancers. Targeted inhibition of mRNA by miRNA can be hindered by competing endogenous RNAs (ceRNAs), such as circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) [6]. miR-944 is regulated by eleven ceRNAs, including six circRNAs and five lncRNAs. miR-944 can target and suppress 27 protein-coding genes, thereby regulating cancer cell behaviors such as cancer cell cycle, We downloaded the TCGA (pan-cancer) dataset from the UCSC Xena database (https: //xenabrowser.net (accessed on 20 June 2022)). We extracted miR-944 expression data (RPM) in 33 cancer samples and performed log2(RPM+1) transformation. We excluded cancer types with <3 control samples and finally retrieved miR-944 expression data in 16 TCGA cancer types. We calculated the quantile percentage of miR-944 expression among all non-zero-expressed miRNAs in each of these 16 cancer types. As shown in Figure 1A, miR-944 was highly expressed in nine tumors (0.75-1.0 quantile, Q4), while miR-944 was moderately expressed in other seven tumors (0.5-0.75 quantile, Q3).
We compared differences in miR-944 expression between non-tumor and tumor samples of 16 cancer types (unpaired Wilcoxon test, R version 4.1.3). As shown in Table 2 and Figure 1B, We found that miR-944 was significantly upregulated in five tumors (BLCA, HNSC, LUSC, THCA, and UCEC); significantly downregulated in two tumors (BRCA and PRAD) ( Figure 1B). Notably, our TCGA analysis demonstrated the association of miR-944 expression with cancer risk in bladder cancer, head and neck squamous cell carcinoma (HNSCC), thyroid cancer, and prostate cancer, which has not been reported yet.  The blue font indicates that miR-944 is significantly low expressed in this cancer type; the red font indicates that miR-944 is significantly highly expressed in this cancer type; (B) comparison of miR-944 expression levels between non-tumor and tumor groups in the TCGA database. *** means p < 0.0000625; ** means p < 0.000625; * means p < 0.003125; ns means no significant difference; (C) overview of SNVs of TP63; (D) mutation types in TP63 protein domains in various cancers; (E) differences in the expression level of miR-944 between the TP63 mutant group (Mut) and the wild group (WT). Ns   We also calculated differences in the expression of miR-944 between patients of different genders or races based on the TCGA database. The results were shown in Figure S1. There was no significant difference in the expression level of miR-944 between males and females in cancer. In BLCA and ESCA, the level of miR-944 in whites was significantly lower than that in other races.

Co-Expression of TP63 Transcripts and miR-944
miR-944 is located in the TP63. Previous studies have shown that ∆Np63, but not TAp63, can directly regulate the expression level of miR-944 by recruiting the transcription factor AP-2 [19]. The high correlation between the expression of TAp63 and miR-944 may be due to the common upstream regulators of TAp63 and ∆Np63, resulting in a significant positive correlation between TAp63 and ∆Np63.
In order to explore the relationship between TP63 and miR-944, we obtained the expression data of miR-944, TAp63, and ∆Np63 in TCGA (pan-cancer) from the UCSC Xena database (https://xenabrowser.net (accessed on 20 June 2022)).

TP63 Copy Number Variation (CNV) and miR-944
CNV is a chromosomal structural variation, often caused by genome rearrangement, resulting in duplication, deletion, or copy number change in specific regions of the genome [41]. We downloaded the CNV of the TP63 gene from UCSC Xena (https: //xenabrowser.net/ (accessed on 20 June 2022)). We calculated the correlation of TP63 CNV with TP63, ∆Np63, and miR-944 expression (Pearson's correlation test). As shown in Figure 2, in CESC, ESCA, LUSC, PAAD, and TGCT, the expression level of miR-944 was significantly positively correlated with TP63 CNV (p < 0.0001 and r > 0.3). Notably, in cancers with significantly upregulated miR-944 (BLCA, HNSC, and LUSC), the expression level of miR-944 was at Q4, higher than that of most miRNAs, and miR-944 expression was positively correlated with TP63 CNV. However, in BRCA and PRAD where miR-944 was significantly downregulated, miR-944 expression was not significantly correlated with TP63 CNV.

miR-944 and Cancer Cell Behaviors
As shown in Table 3 and Figure 3, miR-944 can inhibit 27 protein-coding genes, thereby regulating various behaviors of cancer cells, such as cell cycle, proliferation, EMT, apoptosis, invasion, and migration.

Regulatory Effect of miR-944 on Cell Cycle
The cell cycle, a continual event in which a cell replicates its genetic material, grows, and divides into two daughter cells, is a ubiquitous and tightly regulated process [46]. Checkpoints at various stages of the cell cycle transmit abnormal signals to effectors that trigger cell cycle arrest [47].
In non-small-cell lung cancer (NSCLC), the CircFUT8/miR-944/YES1 axis can increase the proportion of cells in the G0/G1 phase of H522 and H1975 cell lines, reduce the proportion of cells in the S phase, and inhibit the cell cycle [15]. In CRC, miR-944 can block cell cycle G1 phase progression in the HCT116 cell line by targeting COP1 and MDM2 [37]. In EC, miR-944 downregulated CADM2 to promote cell cycle progression in two EC cell lines (Ishikawa and KLE), while miR-944 knockdown resulted in cell cycle arrest in the G1 phase [22].

Regulatory Effect of miR-944 on EMT
EMT is a biological process in which epithelial cells acquire mesenchymal characteristics. EMT is closely related to the occurrence, development, and recurrence of tumors [50]. miR-944 inhibits EMT progression in cancer cells by targeting two genes (MACC1 [29,35] and GATA6 [38]). miR-944 can also inhibit the EMT process of cancer cells by participating in the SNHG6/miR-944/RAB11A axis [45].

Exogenous Regulators of miR-944
As shown in Figure 5B, studies have shown that exogenous factors such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetaldehyde, alcohol, and human papillomavirus (HPV) can affect the expression of miR-944 in vivo and play a role in the occurrence and development of cancer.
NNK is the main component of tobacco extract [44]. CISH contains an SH2 domain and a SOCS box domain that endogenously represses STAT [44]. NNK upregulates the expression level of miR-944 in OSCC cell lines (OEC-M1 and SCC-25), thereby increasing the targeted inhibition of CISH by miR-944, which in turn impedes the activation of the Jak/STAT3 signaling pathway and promotes Inflammation [44]. Acetaldehyde and alcohol can upregulate the expression of miR-944 in HCC, which in turn increases the sensitivity of HCC cells to doxorubicin [36].
We also calculated the effect of tobacco and alcohol history on the expression level of miR-944 in patients based on the TCGA database. The results were shown in Figure S2. In LUAD, the expression level of miR-944 in light smoking patients was significantly lower than that in non-smoking patients; however, drinking history did not affect the expression level of miR-944 in patients.
HPV is the leading cause of CxCa, which is the fourth leading cause of death in women worldwide [55]. HPV E6/E7 can significantly upregulate the expression level of miR-944 in HPV-infected CxCa tissues, resulting in larger tumors, later international federation of gynecology and obstetrics (FIGO) stage, and a higher level of lymph node metastasis rate [5]. NNK is the main component of tobacco extract [44]. CISH contains an SH2 domain and a SOCS box domain that endogenously represses STAT [44]. NNK upregulates the expression level of miR-944 in OSCC cell lines (OEC-M1 and SCC-25), thereby increasing the targeted inhibition of CISH by miR-944, which in turn impedes the activation of the Jak/STAT3 signaling pathway and promotes Inflammation [44]. Acetaldehyde and alcohol

miR-944 Is Involved in a Variety of Cancer-Related Signaling Pathways
As shown in Figure 6, miR-944 is involved in three signaling pathways that regulate the occurrence and development of cancer. miR-944 is involved in regulating the Wnt/βcatenin signaling pathway by targeting GATA6 in CRC [8]. miR-944 is involved in regulating the Jak/STAT3 signaling pathway by targeting CISH in OSCC [44], STAT1 in LUAD [30], and SOCS4 in LUSC [21]. In the PI3K/AKT signaling pathway, miR-944 targets VEGF in osteosarcoma (SaOS) [39], MACC1 in GC [35], and MDM2 in NPC [7] and CRC [37]. Figure 6. "↑": activation of the signaling pathway will promote the cell behavior; "↓": activation of the signaling pathway will inhibit the cell behavior. miR-944 is involved in the regulation of three Figure 6. "↑": activation of the signaling pathway will promote the cell behavior; "↓": activation of the signaling pathway will inhibit the cell behavior. miR-944 is involved in the regulation of three signaling pathways. miR-944 plays a regulatory role in the occurrence and development of cancer by participating in the Wnt/β-catenin, PI3K/AKT, and Jak/STAT signaling pathways.

miR-944 and the Wnt/β-Catenin Pathway
The Wnt/β-catenin signaling pathway drives tumor cell proliferation, EMT, invasion, and migration, and plays an important role in tumor recurrence and metastasis [56].
GATA6 can promote the accumulation of β-catenin in the cytoplasm by enhancing EGF signaling or increasing the level of intracytoplasmic calcium [8]. After entering the nucleus, free β-catenin binds to transcription factors of the TCF/LEF family, activates the Wnt/β-catenin pathway, and initiates transcription of downstream genes [57]. miR-944 inhibits the activation of the Wnt/β-catenin signaling pathway by targeting GATA6, thereby inhibiting the EGF-induced EMT process in CRC cancer cell lines (HCT116 and SW480) [8].

miR-944 and the Jak/STAT Pathway
The Jak/STAT signaling pathway is involved in many biological processes that promote tumor cell proliferation, survival, invasion, and migration [58]. The Jak/STAT signaling pathway is overactivated in most cancers and is often associated with poor clinical prognosis [58]. STAT1 and STAT3 are important members of the STAT family, both of which can promote cell survival and induce immune tolerance [59]. CISH is a negative regulator of endogenous STAT3 signaling [44]. In OSCC, miR-944 can target and inhibit the expression level of CISH, promote the activation of the Jak/STAT signaling pathway, and then promote the occurrence of inflammation and the invasion and migration of cancer cells [44]. In LUAD, miR-944 directly targets and inhibits STAT1 expression, inhibits the activation of the Jak/STAT signaling pathway, thereby inhibiting cancer cell proliferation, and also hinders tumor growth in BALB/c nude mice [30].
SOCS4 can inactivate Jak, thereby blocking the activation of the Jak/STAT signaling pathway [60]. In LUSC, miR-944 targets and inhibits SOCS4, which in turn activates the Jak/STAT signaling pathway and promotes the growth and proliferation of cancer cells [21].

miR-944 and the PI3K/AKT Pathway
The PI3K/AKT signaling pathway can regulate a variety of key epigenetic modifiers and promote the occurrence and development of cancer [61]. Abnormal activation of the PI3K/AKT signaling pathway can promote the proliferation and metastasis of cancer cells and can also promote angiogenesis and induce drug resistance in cancer cells [62].
VEGF can activate Src and PI3K in turn by binding to membrane receptors [63]. PI3K promotes the phosphorylation of PIP2 to generate PIP3, which recruits and activates AKT at the plasma membrane, thereby activating the PI3K/AKT signaling pathway [64]. In SaOS, low expression of miR-944 can upregulate VEGF expression, activate PI3K/AKT signaling pathway, and promote cancer cell proliferation [39].
MACC1 can activate the PI3K/AKT signaling pathway by increasing c-MET levels [65]. In GC, under-expressed miR-944 upregulates MACC1 expression and activates the PI3K/AKT signaling pathway to promote cancer cell invasion and migration [35].
MDM2 inhibits the activity of the transcription factor p53 and degrades it by ubiquitination [66]. In NPC [7] and CRC [37], low expression of miR-944 upregulates MDM2 expression, inhibits p53 transcriptional activity, and promotes the cell cycle of cancer cells.

Prognostic Value of miR-944
The dysregulation of miR-944 is not only closely related to the pathological status of cancer tissues but also significantly related to the diagnosis of cancer risk and the prognosis of patients. As shown in Table 5, in CRC, HCC, NPC, and BrC, low expression of miR-944 was closely associated with poor patient prognosis. In CxCa, low expression of miR-944 was associated with better patient prognosis.
In NPC, low expression of miR-944 was associated with advanced clinical stage and was significantly associated with shorter OS [7]. In CRC, low expression of miR-944 was associated with advanced tumor invasion stage, lymph node and distant metastasis stage, TNM stage, more liver metastasis, and shorter OS and progression-free survival (PFS) [8,9,37]. In BrC, low expression of miR-944 was associated with advanced clinicalstage, late TNM stage, and shorter OS [10,67].
In LUAD, STAT1 is a downstream target of miR-944, and STAT1 expression is negatively correlated with miR-944 and significantly associated with shorter OS [30]. In addition, ceRNAs of miR-944 were highly expressed in four cancers including NPC, GC, NSCLC, and TSCC, and they could significantly suppress miR-944 expression and were associated with a poorer prognosis in cancer patients. In NPC, highly expressed circSERPINA3 inhibited the miR-944 expression and was significantly associated with later clinical stage and shorter OS [7]. In GC, highly expressed circHAS2 was negatively correlated with miR-944 and significantly correlated with later T stage, later TNM stage, more aggressive lymph node metastasis and neural/vascular invasion, and shorter OS [34]. In NSCLC, high expression of SNHG6 was negatively correlated with miR-944 and was significantly associated with later TNM stage, larger tumor size, and shorter OS [32]. In TSCC, high expression of PRNCR1 was negatively correlated with miR-944 and was significantly associated with larger tumor size, later clinical stage, more lymph node metastasis, and shorter OS [33]. We used CancerMIRNome (http://bioinfo.jialab-ucr.org/CancerMIRNome/ (accessed on 25 August 2022)) to explore the difference in the prognosis of patients with high and low expression of miR-944 in the TCGA database (PMID: 34500460). The results were shown in Figure S3. In BLCA, patients with high expression of miR-944 had better OS, while patients with high expression of miR-944 in LGG and THYM had poor prognoses (p < 0.05).

miR-944 and Its Therapeutic Value
Studies have shown that miRNAs are promising as novel tools or therapeutic targets for cancer therapy [68]. In five cancers (BrC, NSCLC, T-cell acute lymphoblastic leukemia (T-ALL), CRC, and CxCa), miR-944 was significantly associated with drug resistance in cancer cells. In NSCLC and HNSCC, miR-944 is involved in the molecular mechanism of action of two anticancer drugs.

miR-944 and Drug Resistance of Cancer Cells
Drug resistance of cancer cells is one of the important reasons for chemotherapy failure [69]. As shown in Figure 7A, miR-944 is involved in the resistance of cancer cells to various anticancer drugs.
DDP is a platinum-based anticancer drug widely used in the treatment of various solid cancers [70]. DDP induces apoptosis by interacting with purine bases on DNA to generate DNA damage [71]. In BrC, highly expressed miR-944 inhibits the BINP3/MMP/caspase3 axis, resulting in DDP resistance in MCF-7 cells [26]. In NSCLC, high expression of CircZFR activates the CircZFR/miR-944/LASP1 axis, resulting in DDP resistance in A549 and H1299 cell lines [11].  RAPA is a macrolide that specifically inhibits mTOR [72,73]. The mTOR signaling pathway is a major regulator of cell growth and metabolism, and its abnormal expression can induce a variety of human diseases, such as cancer, diabetes, and neurological diseases [74]. In T-ALL, miR-944 inhibits the THBS1/MMP2 axis, thereby inducing resistance to RAPA in Molt-4 cells [12].
DOX is an anthracycline antibiotic commonly used in the treatment of breast cancer, Hodgkin's disease, and lymphoblastic leukemia [75]. DOX can induce apoptosis of cancer cells by regulating cell autophagy, but it also has certain toxicity to other organs, such as the heart [76]. In CRC, activation of the CircCSPP1/miR-944/FZD7 axis leads to elevated levels of downstream p-gp, LRP, and MRP, which in turn lead to resistance to DOX in LoVo and HCT116 cells [13].
PTX, a member of the paclitaxel family, can block mitosis and cause apoptosis [77]. IL10 has immunosuppressive and tumor-promoting abilities [78]. In CxCa, activation of the CircZFR/miR-944/IL10 axis leads to resistance to PTX in HeLa and SiHa cells [14].

miR-944 and Drug Therapy
As shown in Figure 5A, miR-944 was associated with the molecular mechanism of two anticancer drugs (matrine and 188Re-liposome).
188Re-liposome is a radioactive nanoparticle that emits γ and β rays, which can be used for imaging validation and cancer therapy, respectively. 188Re-liposome can be preferentially distributed in tumors, and its anticancer effect is currently in clinical trials [80]. In HNSCC, 188Re-liposome can downregulate miR-944, thereby inhibiting tumor growth [16].

Discussion
miR-944 is located in the fourth intron of TP63. An in-depth study of the relationship between miR-944 and TP63 and ∆Np63 can broaden the understanding of the molecular mechanism of miR-944 in cancer. In the current study, the abnormal expression pattern of miR-944 in most cancers has been roughly clear. However, further study of the abnormal expression patterns of miR-944 in different BrC subtypes is required in BrC. Furthermore, we can infer that TP63 SNV has no significant effect on the expression level of miR-944, while TP63 CNV has a significant effect on the expression level of miR-944, mainly in squamous cell carcinoma.
The expression of miR-944 levels in BrC is still controversial. It was found that the expression level of miR-944 was lower in BrC cell lines MDA-MB-231, MCF-7, MDA-MB-453, ZR-75, and T47-D than in non-cancer cell line MCF-10A. The expression of miR-944 was lower in locally invasive breast tumor tissues from BrC patients than in adjacent tissues [27]. However, some studies have found that the level of miR-944 in cancer tissues and serum of BrC patients is upregulated compared with the serum of adjacent tissues and normal people [26]. These differences may arise from tissue heterogeneity. Further in-depth study of the expression levels of miR-944 in different cancer subtypes is required. Previous TCGA database analysis used the optimal cut-off method of miR-944 expression level to predict the OS level of patients and found that high levels of miR-944 were associated with longer OS in patients [10,67]. However, in this study, using the median truncation method of miR-944 expression levels, we found that there was no significant difference in OS levels between the high and low groups ( Figure S3). In conclusion, more clinical data in the future are needed to confirm the prognostic value of miR-944.
In TCGA-BRCA, the expression level of miR-944 in cancer tissues was significantly lower than that in non-cancer tissues, and was significantly correlated with ∆Np63 and TAp63 expression, but not with TP63 CNV. There are multiple subtypes of BrC, and the inconsistency of the abnormal expression pattern of miR-944 in breast cancer may be related to different BrC subtypes. In addition, miR-944 was significantly underexpressed in LUAD tissues and cell lines (such as A549 and H1299), and significantly overexpressed in LUSC tissues (including TCGA-LUSC). Likewise, miR-944 was significantly underexpressed in the tissues and serum of patients with esophageal adenocarcinoma, whereas it was highly expressed in TCGA-ESCA. miR-944 expression was upregulated when cancer cells were derived from highly keratinized cells [17]. During epidermal differentiation, ∆Np63 can upregulate the expression of miR-944 [17], which may be the reason for the difference in the abnormal expression pattern of miR-944 between squamous cell carcinoma and adenocarcinoma.
In CRC, HCC, NPC, and BrC, low levels of miR-944 can serve as a biomarker for poor clinicopathological features and poor prognosis in cancer patients. In CxCa, however, low levels of miR-944 could serve as a biomarker for better clinicopathological features and favorable prognosis in cancer patients. Among the ceRNAs of miR-944, highly expressed circSERPINA3, circHAS2, SNHG6, and PRNCR1 can be used as biomarkers for poor clinicopathological features and poor prognosis in cancer patients. In addition, highly expressed STAT1, a target PCG of miR-944, can serve as a biomarker for poor clinicopathological features and poor prognosis in cancer patients. The above evidence suggests that miR-944 has the potential as a prognostic marker for cancer.
There are still many deficiencies in the research of miR-944. In future studies, the association of miR-944 with resistance to more anticancer drugs needs to be investigated, as well as the relationship between the abnormal expression of miR-944 in cancer and the efficacy of drug treatment.

Conclusions
This work provides a systematic review of miR-944, points out the potential of miR-944 to become a hot spot in cancer research, and provides potential clues and directions for the follow-up research of miR-944. The bioinformatics analysis herein revealed the potential value of miR-944 as a biomarker for squamous cell carcinoma. At the same time, this paper also points out some controversies and deficiencies in the current research on miR-944. Future research on miR-944 could focus on the molecular regulation of miR-944 dysregulation and its molecular mechanisms associated with antitumor drug resistance and efficacy. This will lay a theoretical foundation for the clinical application of miR-944 in tumors.