Most miRNAs regulate cell cycle by inducing G0/G1 phase arrest. E2F transcription factors, cyclins and CDKs families play significant roles in cell cycle regulation (Huber et al., 2021). It can be seen from literatures that miRNAs induce G0/G1 phase arrest mainly by influence the expression of cell cycle regulators directly or indirectly. In cases of human glioma, overexpression of miR-320c dramatically down-regulated the ratio of S phase cells and blocked cell cycle at G1 phase by suppressing the expression of cyclin D1 and CDK6 (Lv et al., 2018). MiR-424-5p repressed cell proliferation and induced G0/G1 phase arrest by directly regulating its downstream gene E2F7 in hepatocellular carcinoma cells (Zhao et al., 2020). Moreover, Zhang et al.(Zhang et al., 2018) proved that miR-1258 was inhibited in colorectal cancer, and upregulation of miR-1258 blocked cell cycle at G1 phase in vivo and in vitro. Subsequently, miR-1258 was found to affect the expression of cyclin D1 and p21 through miR-1258s target E2F8 (Zhang et al., 2018). These results confirmed that miRNAs might influence multiple E2F transcription factor family members, thereby affecting the level of cyclins and CDK inhibitors and ultimately regulating G0/G1 phase. It is generally recognized that CDKs are cyclin-dependent kinases and affected by cyclins or CDK inhibitors such as p21. But surprisingly, the expression of CDK2 was not changed with cyclin D1 and p21 in colorectal cancer (Zhang et al., 2018). Therefore, more cell cycle-related CDKs expression should be detected to further determine that which CDK was indirectly affected by miR-1258 in colorectal cancer.

In addition to the typical cell cycle regulators mentioned above, some novel proteins have also been found to participate in the regulation of G0/G1 phase by miRNAs, enriching the regulatory network of miRNAs on cell cycle. For instance, triple negative breast cancer cells expressed high level of Neuropilin-1 (NRP-1) (Zhang et al., 2021). MiR-124-3p bound to 3′-UTR region of NRP-1 mRNA, blocked cell cycle at G0/G1 phase and inhibited cell proliferation (Zhang et al., 2021). After that, knockdown of NRP-1 increased the level of p27 and suppressed cyclin E and CDK2 (Zhang et al., 2021). It is not difficult to conjecture that the novel proteins may act as mediators between miRNAs and cell cycle, and affect the downstream cyclins, CDK inhibitors and CDKs family. Besides, kinesin family protein 22 (KIF22) is a kallikin-like DNA-binding protein that play a role in tumorigenesis (Wang et al., 2021). The KIF22 silencing resulted in an increase of G0/G1 phase esophageal squamous cells (Wang et al., 2021), leading to further study on the correlation between KIF22 and cell cycle. The binding of miR-122 to KIF22 was found and inhibition of miR-122 blocked the regulatory effect of si-KIF22 on p21, p27, cyclin D1 and CDK2 (Wang et al., 2021), suggested that these were all indirect downstream proteins of miR-122. Furthermore, chaperonin containing TCP1 subunit 3 (CCT3) is a component of chaperonin in eukaryotes, which has an impact on cell proliferation (Temiz et al., 2021). The mimic transfection of miR-24-3p, miR-128-3p, and miR-149-5p suppressed the level of CCT3 in ductal-breast adenocarcinoma and prostate carcinoma cells, which induced G0/G1 phase arrest and disturbed the mitochondrial membrane potential, eventually triggering apoptosis (Temiz et al., 2021). This result implied that three miRNAs above could not completely repair the DNA damages, hence inducing apoptosis. In summary, NRP-1 and KIF22 act as the novel downstream proteins of miRNA, and CCT3 is at least affected by miRNA, thus affecting the G0/G1 phase.

Large numbers of miRNAs induce G1 phase arrest, whereas only a few miRNAs promote cell cycle by inducing G1 phase progression, which possibly because cells tend to suspend the cell cycle to prevent disordered cell replication when DDR occurs. For example, high concentration of mmu-miR-762 inhibitor arrested the cell cycle of mouse colon cancer cells at G0/G1 phase (Lai et al., 2021), suggesting that mmu-miR-762 might induce G1 phase progression. Zhao et al. (2019) found that under the infection of herpes simplex virus 2, miR-H4-5p bound to 3′-UTR of CDK inhibitor 2A and cyclin-dependent kinase-like 2 for negative regulation, to promote HeLa cells of S phase to the ratio of 41.8%, which was significantly higher than that in HeLa cells in control. In another study, transcriptomic sequencing of reticuloendotheliosis virus infected chicken embryo fibroblasts found that cyclin D1-CDK6 complex played a key role in the transition process from G1 phase to S phase (Zhai et al., 2018). KEGG enrichment analysis showed that target genes of a new miRNA, novel-72 was found to promote cell cycle and cell proliferation in this virus-infected cells (Zhai et al., 2018). From the reports so far, miRNAs might promote the transition from G1 phase to S phase in cells under the infection by virus, but more studies are needed to further elucidate that if the conjecture can be applied to more viruses.

A variety of miRNAs affect cell cycle by inducing G2 phase arrest, whereas whether miRNAs can promote G2 to M phase transition is rarely reported and not clear. MiR-191 has been reported increased in HeLa cells treated with cisplatin, an effective chemotherapeutic agent that induced DDR (Xu et al., 2020). Overexpression of miR-191 induced G2/M arrest via targeting chromosome condensation 2 regulator (RCC2). The inhibition of RCC2 mimicked the effects of miR-191 (Xu et al., 2020), which clarified the mechanism of miR-191 on cell cycle. Compared with that in healthy people, the level of miR-582-3p was decreased in blood of leukemia patients, and overexpression of miR-582-3p increased the ratio of G2/M phase cells in acute myeloid leukemia (Li et al., 2019). Cyclin B2 encoded by CCNB2 gene is a regulator of G2/M transition (Li et al., 2019). MiR-582-3p reduced the level of cyclin B2 via binding to CCNB2 mRNA, accompanied by its negative effects on CDK1 and cyclin B1 expression (Li et al., 2019). Similarly, miR-181c-5p blocked cell cycle at G2/M phase in injured osteoblasts via binding to 3′-UTR of cyclin B1 (Sun Z. et al., 2019). These studies confirmed that miRNAs regulated the proteins associated with G2 to M phase transition, including RCC2, cyclin B1 and cyclin B2. Moreover, a combinatorial treatment of miR-143 and miR-506 made cell cycle halt at G2/M phase transitions and suppressed the levels of CDK1, CDK4 and CDK6 in A549 non-small lung cancer cells (Hossian et al., 2019), suggested that miR-143 and miR-506 might induce G2 phase arrest via CDKs family, which revealed the potential of multiple miRNAs acting together on cell cycle.

Based on the fact that DNA double-strand damages cause the most serious consequences, most of the current studies on DNA damage repair focus on the repair of DSBs, and a few reports have demonstrated the regulatory role of miRNA in DNA SSBs. Poly (ADP-ribose) polymerase 1 (PARP-1) is a crucial DNA repair enzyme deeply involved in SSBs, and also related to DSBs (Yakovlev, 2015). The loss of PARP-1 activity leads to the SSBs accumulation, and SSBs may be converted to DSBs subsequently (Yakovlev, 2015). In human neuroblastoma cells, acute and chronic cocaine treatment decreased the level of miR-125b and increased the expression abundance of PARP-1, but did not change the level of cleaved-PARP-1 (Dash et al., 2017), implied that miR-125b was probably associated with DNA damage repair, and might have little effect on apoptosis marked by cleaved-PPAR-1. After that, the assays confirmed that miR-125b bound to 3′-UTR of PARP-1 mRNA (Dash et al., 2017). A few years later, another study used the luciferase reporter and pulldown assays found that miR-124 also directly bound to 3′-UTR of PARP-1 mRNA (Dash et al., 2020). Interestingly, the binding region of miR-124 and PARP-1 was partially same as the target sequence of miR-125b on PARP-1 (Dash et al., 2020), which was well demonstrated that a target gene can be regulated by several different miRNAs. Next, the titration experiment was carried out to assess the binding activity of two miRNAs to PARP-1, and showed that compared with miR-125b, miR-124 conferred a dominant post-transcriptional inhibition on PARP-1 (Dash et al., 2020). Therefore, in view of the same binding region, further investigation is needed to clarify that whether the sequence is essential for a miRNA binding with PARP-1 and whether other miRNAs containing this sequence have a negative effect on PARP-1. Subsequently, this sequence may serve as a novel and effective characteristic for screening for DNA SSBs related regulatory miRNAs. In addition to affecting PARP-1, miRNAs may also interfere with DNA SSBs in other way. Kang et al. (2019) found that miR-3912-5p might bind to 3′-UTR of BER protein NEIL2, thereby affecting DNA damage in lens cells. And miR-590 slowed the proliferation and promoted the repair of SSBs in embryonic stem cells (Liu et al., 2014), but the mechanism of which is still unclear.

DSB is the most serious type of DNA damage, and its excessive accumulation leads to the genomic instability and may result in tumorigenesis. The formation of DSBs is often accompanied by phosphorylation of histone H2AX into γ-H2AX (Zeng et al., 2019). The way to repair DSBs depends on the cell cycle: NHEJ fixes DSBs mainly in G0/G1 phase, which is usually faster but prone to errors (Srinivasan et al., 2019). HR fixes DSBs in S and G2/M phases, which is slow and not prone to errors (Yakovlev, 2015; Piotto et al., 2018). In miR-34a-deficient hematopoietic stem cells, the content of γ-H2AX was increased, whereas the expression of DNA repair genes associated with HR and NHEJ was decreased (Zeng et al., 2019), determined that miR-34a had a positive effect on DDR. C-terminal binding protein interaction protein (CTIP) is an endonuclease, whose function is to recognizes damaged DNA specifically and participates in the HR process (Yang et al., 2019). The report showed that miR-130b facilitated the production of DSBs and subsequently promoted apoptosis by targeting CTIP in cervical cancer cells (Yang et al., 2019). It is commonly believed that BRCA1 and RAD17 proteins are associated with DDR, and the transcription inhibition of them leads to excessive build-up of DNA DSBs (Valenti et al., 2019). Experiments verified that the miR-205-5p/BRCA1/RAD17 axis increased the level of γ-H2AX and made DNA repair inefficient in head and neck squamous cell carcinomas in vivo and in vitro (Foray et al., 2003; Valenti et al., 2019), revealed that miR-205-5p/BRCA1/RAD17 axis had the potential to be a therapeutic target for cancer. Meanwhile, the value of miR-205-5p as a potential therapeutic agent needs to be further confirmed.

RAD51 is an extremely important HR regulator, which can be an indirect or direct downstream molecule of miRNAs to influence DNA DSBs repair. BRCA1 was found to directly bind to RAD51 (Zhao et al., 2017), which might be the upstream factor of RAD51 in DDR process. Meanwhile, RAD51 was negatively regulated by RING1 protein, which was a target of miR-3909 (Han et al., 2020). Moreover, all miR-34 family (miR-34a, miR-34b and miR-34c) could suppress the expression of RAD51 to inhibit HR process in human colon adenocarcinoma cells (Chen S. et al., 2019). This result provides solid evidence that the effects of miRNAs on DDR depend largely on the structure of miRNAs. Furthermore, Piotto et al. (2018) found that miR-96-5p targeted RAD51, whereas miR-874-3p targeted NHEJ related genes (PRKDC, LIG1) and affected the expression abundance of DNA-PKcs, leading to death of non-small lung cancer cells. The effects of miR-96-5p and miR-874-3p on DNA repair were similar to chemical inhibitors of HR and NHEJ process respectively (Piotto et al., 2018), which perfectly confirmed that miR-96-5p and miR-874-3p have their respective roles in the repair of DNA DSBs. In addition, γ-ray irradiation increased the level of miR-1246 in exosomes and treating unirradiated cells with this exosome reduced the activity of NHEJ via miR-1246′ binding with 3′-UTR of LIG4 (Mo et al., 2018). The result confirmed that exogenous stimulation could alter the content of a miRNA in exosomes, and raised the possibility that the delivery of miRNAs by exosomes might be a potential therapeutic approach for diseases by influencing the DDR.

In previous studies, the positive effects of miRNAs on apoptosis have been reported, and the downstream genes of miRNAs include BCL-2 family (pro-apoptotic gene Bax, Bim and anti-apoptotic gene Bcl-2, etc.), Fas-associated phosphatase 1 (Fap-1), Caspase family and new molecules associated with apoptosis. In prostate cancer cells, overexpression of miR-29b upregulated the expression of Bim, induced the pro-apoptotic factor cytochrome C and cleaved-PARP-1 release, leading to apoptosis in a time-dependent manner (Agarwal et al., 2009; Sur et al., 2019). Death receptor-mediated apoptosis pathway is activated by Fas interacting with Fas-associated protein with death domain (FADD). After that, the Caspases family was activated (Pozzesi et al., 2014). MiR-200c-3p was believed to link with endothelial function via its target Fap-1, and Fap-1 is an inhibitor of Fas (Jiang et al., 2020). As a result of miR-200c-3p overexpression, Fap-1 was inhibited and Fas was activated, leading to increased FADD, cleaved-caspase-8 and cleaved-caspase-3. These effects were abrogated by miR-200c-3p inhibitor alone or co-use of miR-200c-3p inhibitor and siRNA of Fap-1 (Jiang et al., 2020), which is well confirmed that miR-200c-3p significantly promoted the death receptor-mediated apoptosis pathway.

A recent study showed that the expression of miR-181c-5p was up-regulated in the early stage of myocardial infarction, whose target gene is protein tyrosine phosphatase nonreceptor type 4 (PTPN4) (Ge et al., 2019). As a result, PTPN4 silencing elevated the Bax/Bcl-2 ratio, the abundance of cleaved-caspases 3 and produced more TUNEL positive cells in cardiomyocytes, which exacerbated mitochondria-mediated apoptosis and mimicking the pro-apoptotic effect of miR-181c-5p overexpression (Ge et al., 2019), confirming that PTPN4 is a new molecule involved in apoptosis. As mentioned above, CCT3 is a target of miR-24-3p and miR-128-3p. In ductal-breast adenocarcinoma cells, the expression of BAX was increased via suppressing CCT3 by miR-24-3p or miR-128-3p (Temiz et al., 2021). Besides, miR-24-3p transfection induced the release of cytochrome C and then enhanced the activity of initiator and effector caspase (caspase 9 and 3). And miR-128-3p did not affect cytochrome C release, but increased the proportion of late apoptotic cells and also enhanced the caspase activity (caspase 8,9 and 3) (Temiz et al., 2021). Therefore, miR-128-3p may have stronger binding activity to CCT3 gene than miR-24-3p, thus leading to more characteristics of late apoptosis. In this study, miR-128-3p influenced BAX and BCL-2 located in mitochondria, as well as caspase 8 in the exogenous apoptosis pathway, demonstrating that miR-128-3p simultaneously regulate both mitochondria-dependent and mitochondria-independent apoptosis. To sum up, several miRNAs affect apoptotic participants and promote apoptosis by targeting Fap-1, PTPN4 and CCT3. But most of the published reports only found the causal relationship between miRNAs and apoptosis, and the specific targets of miRNAs involved in apoptosis need to be further investigated.

The miRNAs not only take part in the promotion of apoptosis, but also have a link with the inhibition of apoptosis. PDCD4 is a tumor suppressor, and its knockdown activated the transcription factor AP-1 (Song et al., 2019). MiR-21 directly inhibited apoptosis by the miR-21/PDCD4/AP-1 axis in infarcted cardiomyocytes (Song et al., 2019). Besides, the application of exogenous miR-21 significantly reduced apoptosis and improved cardiac function in preclinical myocardial infarction mice (Song et al., 2019), suggested that miR-21 might be a potential agent to treat myocardial infarction. Similarly, the expression of miR-21 was decreased in myocardial injured rats. Overexpression of miR-21 reduced the Bax/Bcl-2 ratio and the level of caspase-3 by inhibiting TLR4/nuclear factor-κB (NF-κB) pathway (Pan et al., 2018), leading to the reduced level of apoptosis in rat. Moreover, miR-26a-5p activated the PI3K/protein kinase B (AKT) pathway via targeting phosphate and tensin homology deleted on chromosome 10, which inhibited apoptosis of endothelial cells in coronary artery mice (Jing et al., 2019). Furthermore, the expression of miR-27a was suppressed in vascular endothelium. Inhibition of miR-27a promoted the expression of FADD (Sun Y. et al., 2019), confirming that miR-27a inhibited apoptosis mediated by death receptor pathway. Subsequently, luciferase reporter assay verified that miR-27a inhibited apoptosis via binding to 3′-UTR of FADD (Sun Y. et al., 2019). In summary, multiple signaling pathways are associated with the inhibition of apoptosis by miRNAs, such as TLR4/NF-κB and PI3K/AKT pathway, but the targets of miRNAs among the process have not been elucidated yet.

Some miRNAs play dual roles in the process of apoptosis, and the effect of a miRNA on apoptosis may be cell-type specific or disease-specific. MiR-7 exerts dual effects on apoptosis in different tissues under stress conditions. There is a report showed that miR-7 inhibited mitogen-activated protein kinase kinase (MAPKK)/extracellular regulated protein kinase (ERK) and NF-κB pathways by directly targeting MAPKK kinase 9, leading to the upregulation of apoptosis in pancreatic cancer cells (Xia et al., 2018). Another report of benign prostatic hyperplasia epithelial cells and human prostate epithelial cells also suggested that miR-7 had a promoting effect on apoptosis which was related to PI3K/AKT and Ras-associated factor-1/ERK 1/2 pathways (Han et al., 2019). However, miR-7 inhibitor increased apoptosis in colorectal cancer cells (Mo et al., 2019), suggested that miR-7 might have a negative effect on apoptosis. As mentioned above, miR-181c-5p elevated the level of Bax/Bcl-2 ratio and cleaved-caspases 3, showed a pro-apoptotic effect in cardiomyocytes (Ge et al., 2019). Nevertheless, miR-181c-5p acted on 3′-UTR region of HMGB1 mRNA and inhibited apoptosis in microglia (Li et al., 2021). Compared with miR-181c-5p treatment, the overexpression of HMGB1 showed the adverse effect and enhanced apoptosis (Li et al., 2021), confirming the anti-apoptotic role of miR-181c-5p. From the above, MAPKK/ERK, NF-κB, PI3K/AKT and Ras-associated factor-1/ERK 1/2 signaling pathways are all linked with the regulation of miR-7 on apoptosis. These opposite effects of miR-7 and miR-181c-5p may be caused by differences in cell type and organ function.