MiR-26a-1 Promotes DNA Damage Repair by Inhibiting Sirt1 and KDM5A in Human Liver Cancer Stem Cells

Background: Although miR-26a-1 was down-regulated expressin in several cancers, the role of miR-26a-1in malignancies has yet to be systematically elucidated. Methods: RT-PCR, Western blotting and tumorigenesis test in vitro and in vivo were performed to analyze the signaling pathway. Results: miR-26a-1 inhibits the NAD(+)-dependent deacetylase Sirt1 expression by targeting the 3' noncoding region of Sirt1 which enhances the acetylation modication of H4 on the 16th lysine of histone and the expression of protein arginine methyltransferase PRMT6. Therefore, miR-26a-1 promotes arginine methylation modication of POLB (R137) and histone. On the other hand, miR-26a-1 inhibits the expression of KDM5A by targeting its 3' non-coding region, which enhances the methylation modication of histone H3 ysine 4. Moreover, miR-26a-1 enhances the expression of histone methyltransferase SETD2 dependent on H3K4me3 and further increases the trimethylation modication of the histone H3 lysine 36 . Signicantly, miR-26a-1 promotes the formation of DNA damage repair complex (Rad51-PARP1-ATR-ATM-hMSH6-XRCC-POLB-SKP2) via H3K36me3. In particular, it was found that miR-26a-1 inhibited the function of long non-coding RNA HULC and promoted the formation of DNA damage repair complex. Furthermore, miR-26a-1 promotes the DNA damage repair ability by promoting the DNA damage repair complex to bind to the DNA damage site, thereby inhibiting the DNA damage of liver cancer stem cells. In particular, miR-26a-1 enhanced the binding of H3F3A to Skp2, CUL1, and F-box at the DNA damage site and enhanced the protein ubiquitination modication of H3F3A, which promoted Histone H3 replaces H3F3A by degrading H3F3A, realizing the renewal of histones after DNA damage repair. It was further found that miR-26a-1 inhibited the formation and instability of DNA microsatellites by promoting DNA damage repair, thereby affecting the expression of several cyclins and protein kinases in liver cancer stem cells, such as, inhibiting CDK2 and CyclinE , CDK4, CyclinD1, CDK6, CDK8, CyclinM2, CDK15, pRB, PCNA, MAP3K2, PGK1 and promoting RB, P18, P21/WAF1/Cip1, and thus inhibited the growth of liver cancer stem cells. Strikingly, the rescued-test further conrmed that excessive Sirt1 and KDM5A abrogated the oncogenic function of miR-26a-1. Conclusions: miR26a-1 may acts as the potential biomarker and therapeutic target for liver cancer.

increased surfactantassociated mRNA and protein expression levels (4). In particular, Expression of miR26a exhibits a negative correlation with HMGA1 and regulates cancer progression by targeting HMGA1 in lung adenocarcinoma cells (5).
DNA damage repair plays a important role in hepatocarcinigenesis. The decision between cell survival and death following DNA damage rests on factors that are involved in DNA damage recognition, as well as on factors involved in the activation of apoptosis, autophagy (6). Deviations in this ne-tuning are known to destabilize cellular metabolic homeostasis, as exempli ed in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability (7). DNA repair factors ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining (8).Cancer chemotherapy and radiotherapy are designed to kill cancer cells mostly by inducing DNA damage. (9).DNA damage repair systems have evolved to act as a genome-wide surveillance mechanism to maintain chromosome integrity and impairment of these systems gives rise to mutations and directly contributes to tumorigenesis (10).
In the study, miR-26a-1 inhibits the expression of NAD+-dependent deacetylase Sirt1 and KDM5A, thereafter, miR-26a-1 promotes DNA damage repair, thereby affecting the expression of some cyclins and protein kinases in liver cancer stem cells. Moreover, excessive Sirt1 and KDM5A abolished the oncogenic functions of miR-26a-1 in liver cancer stem cells. In conclusions, miR26a-1 may acts as the potential biomarker and therapeutic target for liver cancer. We also shed light on the fact that the attenuation of deregulated functioning of miRNA could be a viable approach for cancer treatment. Cell Lines Cells were maintained in Dulbecco's modi ed Eagle medium(Gibco BRL Life Technologies) supplemented with 10% heat-inactivated (56ºC, 30 minutes) fetal bovine serum (sigma) in a humidi ed atmosphere of 5% CO 2 incubator at 37ºC.

Materials And Methods
RT-PCR cDNA was prepared by using oligonucleotide (dT), random primers, and a SuperScript First-Strand Synthesis System (Invitrogen). PCR analysis was performed according to the manufacturer. β-actin was used as an internal control.
Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with antibody(appropriate dilution) overnight at 4°C. Signals were visualized by enhanced chemiluminescence plus kit(GE Healthcare).
miR-26a-1 enhances the expression of protein arginine methyltransferase PRMT6 Given that miR-26a-1 increases the acetylation modi cation of histone H4 lysine 16, we will further investigate whether miR-26a-1 enhances protein PRMT6 in liver cancer stem cells. The loading of H4K16Ac with PRMT6 promoter was signi cantly enhanced in the rLV-miR-26a-1 group compared with the rLV group and weakened in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 3A). The binding ability of H4K16Ac with the PRMT6 promoter probe was signi cantly enhanced in the rLV-miR-26a-1 group compared with the rLV group and drcreaed in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 3B). The ability of H4K16Ac and RNA PolII to enter the PRMT6 promoter-enhancer loop was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and weakened in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 3C).
However, it was not signi cantly altered in the rLV-miR-26a-1 + rLV-KDM5A group compared with the rLV group (551892.38 ± 112067.76 vs 677012.65 ± 78394.74, P = 0.06122 > 0.051) (Fig. 6F). The expression of SETD2 was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 6G&H). However, it was not signi cantly altered (Fig. 6I&J). The interaction between histone H3 and SETD2 was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV groupand reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 6K). The H3K36me3 was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 6L). However, it was not signi cantly altered in the rLV-miR-26a-1 + pGFP-V-RS-SETD2 group compared with the rLV group (Fig. 6M). Collectively, these results suggest that miR-26a- Given that miR-26a-1 promotes the trimethylation of histone H3 on lysine 36, we will analyze whether miR-26a-1 to promote DNA damage repair complex formation dependent on the trimethylation of histone H3 on lysine 36. The binding ability of H3K36me3 with Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, and SKP2 was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 7A). The binding of mismatched DNA damage probes to Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 7B). However, the H3K36me3 was signi cantly reduced in the rLV-miR-26a-1 + rLV-KDM4A group compared with the rLV group (Fig. 7C). The binding ability of Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, and SKP2 to mismatched DNA damage probes was not signi cantly altered in the rLV-miR-26a-1 + rLV-KDM4A group compared with the rLV group (Fig. 7D). Next, Transfect the mismatched plasmid was transfected (Fig. 7E) and then perform repeated chromatin immunoprecipitation (CHIP) to analyze the binding ability of the methylated POLB with the mismatched sequence. The results showed that the binding ability of the methylated POLB to the mismatched DNA sequence was signi cantly enhanced in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 7F). The binding ability of mismatched DNA sequences to Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 was signi cantly enhanced in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group ( Fig. 7G). Collectively, these results suggest that miR-26a-1 enhances the binding ability of Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 to mismatch DNA damage dependent on H3K36me3, and promotes the formation of DNA damage repair complexes. [Also see and Supplemental Resuts: miR-26a-1 promotes the formation of DNA damage repair complex dependent on the H3K36me3 (FigureS8A-F)]. miR-26a-1 promotes the formation of DNA damage repair complexes dependent on long non-coding RNA HULC In order to investigate whether miR-26a-1 promotes the formation of DNA damage repair complexes, we rst analyzed whether non-coding RNA HULC is related to the formation of DNA damage repair complexes. HULC was signi cantly increased in the rLV-HULC group compared with the rLV group and reduced in the pGFP-V-RS-HULC group compared with the pGFP-V-RS group (Fig. 8A). The binding ability of PARP1, Rad51 to HULC was signi cantly enhanced in the rLV-HULC group compared with the rLV group and reduced in the pGFP-V-RS-HULC group compared with the pGFP-V-RS group (Fig. 8B). Compared with the rLV group, in the rLV-HULC group, The binding ability of H3K36me3, Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 to mismatched DNA damage probes was signi cantly reduced in the rLV-HULC group compared with the rLV group and enhanced in the pGFP-V-RS-HULC group compared with the pGFP-V-RS group (Fig. 8C). Compared with the rLV group, The binding ability of H3K36me3, Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 to mismatched DNA sequences was signi cantly weakened in the rLV-HULC group compared with the rLV group and increased in the pGFP-V-RS-HULC group compared with the pGFP-V-RS group (Fig. 8D).The mutual binding ability between PARP1, Rad51 to HULC was signi cantly reduced in the rLV-HULC group compared with the rLV group and increased in the pGFP-V-RS-HULC group compared with the pGFP-V-RS group (Fig. 8E).The expression of HULC was not signi cantly altered in the rLV-miR-26a-1 group compared with the rLV group, however, it was signi cantly increased in the rLV-miR-26a-1 + rLV-HULC group (Fig. 8F). The binding capacity of H3K36me3, Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, and SKP2 to mismatched DNA damage probes was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group, however, it was not signi cantly changed in the rLV-miR-26a-1 + rLV-HULC group compared with the rLV group (Fig. 8G). Compared with the rLV group, The ability of Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 to mismatched DNA sequences were signi cantly enhanced in the rLV-miR-26a-1 group compared with the rLV group, however, it was not signi cantly altered in the rLV-miR-26a-1 + rLV-HULC group compared with the rLV group (Fig. 8H). Collectively, these results suggest that that miR-26a-1 promotes the formation of DNA damage repair complexes dependent on long non-coding RNA HULC in liver cancer stem cells.
[Also see and Supplemental Resuts: miR-26a-1 promotes the formation of DNA damage repair complexes dependent on long non-coding RNA HULC (FigureS9A-C)].
The H3F3A ubiquitination modi cation was signi cantly increased in the rLV-miR-26a-1 group in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10C). The ubiquitination level of H3F3A bound by the DNA damage probe was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10D). The H3F3A ubiquitination modi cation was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10E). By transfecting mismatched plasmids, the ubiquitination level of H3F3A bound to the DNA damage probe was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10F). After alisertib was used to induce DNA damage, H3F3A was signi cantly reduced in in the rLV-miR-26a-1 group compared with the rLV group and increased in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10G). The binding ability of H3F3A to the repaired DNA sequence was signi cantly reduced, and the binding ability of Histone H3 to the repaired DNA sequence was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group and reduced in the rLV-Cas9-miR-26a-1 group compared with the rLV-Cas9 group (Fig. 10H). The above results suggest that miR-26a-1 increases the renewal ability of histones after DNA damage repair, that is, enhances the ability of H3F3A to be replaced by Histone H3. H3F3A was signi cantly reduced in the rLV-miR-26a-1 group compared with the rLV group. However, it was not signi cantly altered in the rLV-miR-26a-1 + MG132 group compared with the rLV group (Fig. 10I). The binding ability of H3F3A to the repaired DNA sequence was signi cantly reduced, and the binding ability of Histone H3 to the repaired DNA sequence was signi cantly increased in the rLV-miR-26a-1 group compared with the rLV group. However, it was not signi cantly altered in the rLV-miR-26a-1 + MG132 group compared with the rLV group (Fig. 10J). Collectively, these results suggest that miR-26a-1 increases the renewal ability of histones dependent on protein ubiquitination degradation pathway after DNA damage repair[Also see and Supplemental Resuts: miR-26a-1 increases the renewal ability of histones dependent on protein ubiquitination degradation pathway after DNA damage repair (FigureS11A-N)].

Discussion
In this study, our results suggest that miR-26a-1 inhibits the NAD(+)-dependent deacetylase Sirt1 expression by targeting the 3' non-coding region of Sirt1 which enhances the acetylation modi cation of histone H4 on the 16th lysine and the expression of protein arginine methyltransferase PRMT6.
Therefore, miR-26a-1 promotes arginine methylation modi cation of POLB (R137) and Histone. On the other hand, miR-26a-1 inhibits the expression of KDM5A by targeting its 3' non-coding region, which enhances the methylation modi cation of histone H3 ysine 4. Moreover, miR-26a-1 enhances the expression of histone methyltransferase SETD2 dependent on H3K4me3 and further increases the trimethylation modi cation of the histone H3 lysine 36. Signi cantly, miR-26a-1 promotes the formation of DNA damage repair complex (Rad51-PARP1-ATR-ATM-hMSH6-XRCC-POLB-SKP2) via H3K36me3. In particular, it was found that miR-26a-1 inhibited the function of long non-coding RNA HULC and promoted the formation of DNA damage repair complex. Furthermore, miR-26a-1 promotes the DNA damage repair ability by promoting the DNA damage repair complex to bind to the DNA damage site, thereby inhibiting the DNA damage of liver cancer stem cells. In particular, miR-26a-1 enhanced the binding of H3F3A to Skp2, CUL1, and F-box at the DNA damage site and enhanced the protein ubiquitination modi cation of H3F3A, which promoted the Histone H3 replacng H3F3A, realizing the renewal of histones after DNA damage repair. It was further found that miR-26a-1 inhibited the formation and instability of DNA microsatellites by promoting DNA damage repair, thereby affecting the expression of several cyclins and protein kinases in liver cancer stem cells, such as, inhibiting CDK2 and CyclinE, CDK4, CyclinD1, CDK6, CDK8, CyclinM2, CDK15, pRB, PCNA, MAP3K2, PGK1 and promoting RB, P18, P21/WAF1/Cip1, and thus inhibited the growth of liver cancer stem cells in vivo and in vitro (Fig. 13).
Notably, our results suggest that miR-26a-1 inhibits the growth of human liver cancer stem cells, and excessive Sirt and KDM5A abrogated the oncogenic functions of miR-26-1.Moreover, our results suggest miR-26a-1 targets NAD+-dependent deacetylase Sirt1 and enhances the acetylation modi cation of histone H4 lysine 16.Sirtuin-1 (SIRT1) is a class-III histone deacetylase (HDAC), an NAD+-dependent enzyme deeply involved in gene regulation, genome stability maintenance, apoptosis, autophagy, senescence, proliferation and tumorigenesis. It also has a key role in the epigenetic regulation of tissue homeostasis and many diseases by deacetylating both histone and non-histone target (11)(12)(13).
Accumulating evidence has indicated that SIRT1 is a key regulator of DNA damage and cancer (14,15).
Intriguingly, our results suggest that miR-26a-1 enhances the expression of protein arginine methyltransferase PRMT6 gene dependente on H4K16Ac.Protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and regulates RAS/RAF through CRAF methylation (16). PPARα protects against colon carcinogenesis via regulation of PRMT6 (17). PTEN arginine methylation by PRMT6 suppresses PI3K-AKT signaling (18). CRAF methylation by PRMT6 regulates hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation (19). Furthermore, our results suggest that miR-26a-1 enhances the methylation modi cation of POLB(R137) and histone arginine through PRMT6. Nuclear DNA repair polymerase, POLB, is located in the mitochondria and plays a signi cant role in mitochondrial BER, mtDNA integrity and mitochondrial function (20). Genome instability caused by a germline mutation in the human DNA repair gene POLB (21) Interestingly, our results suggest that miR-26a-1 enhances the methylation modi cation of histone H3 lysine 4 by reducing KDM5A.KDM5A acts as a negative regulator of p53 signaling (22). KDM5A bound directly to MPC-1 promoter region and suppressed the expression (23). KDM5A/5B knockdown resulted in lower viability of HL-60 cells (24). KDM5A acts as a critical editor of the cells' "histone code" that is required to recruit DNA repair complexes to DNA breaks (25). HDAC1 negatively regulates selective mitotic chromatin in a KDM5A-dependent manner (26). Ampli cation of KDM5A is observed in many cancers, including breast cancer, prostate cancer, hepatocellular carcinoma (27). The H3K4 tri-demethylase KDM5A and speci c COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment (28).
Strikingly, our results suggest that miR-26a-1 enhances the binding ability of Rad51, PARP1, ATR, ATM, hMSH6, XRCC5, POLB, SKP2 to mismatch DNA damage dependent on H3K36me3, and promotes the formation of DNA damage repair complexes. RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection (39). PARP1 blockade is synthetically lethal in XRCC1 de cient sporadic epithelial ovarian cancers (40). the ATM, ATR, DNA-PK family proteins can be activated immediately upon DNA damage recognition (41). The phosphorylation of hMSH6 is involved in cellular signaling of either DNA mismatch repair or MMR-dependent damage recognition activities (43). The hMsh2-hMsh6 complex acts in concert with monoubiquitinated PCNA and Pol η in response to oxidative DNA damage in human cells (44). The XRCC genes results in their roles in DNA repair and genetic stability (45). SKP2 promotes tumorigenesis and radiation tolerance through PDCD4 ubiquitination (46). Also, our results suggest that that miR-26a-1 promotes the formation of DNA damage repair complexes dependent on long non-coding RNA HULC in liver cancer stem cells. As an oncogene, HULC promotes tumorigenesis by regulating multiple pathways, such as down-regulation of EEF1E1 (47). LncRNA HULC triggers autophagy via stabilizing Sirt1 (48). Circulating extracellular vesicle-encapsulated HULC is a potential biomarker for human pancreatic cancer (49).
In particular, our results suggest that miR-26a-1 promotes the DNA damage repair by inhibiting Sirt1 and KDM5A. On the other hand, our results suggest that miR-26a-1 increases the renewal ability of histones dependent on protein ubiquitination degradation pathway after DNA damage repair. Distinct H3F3A and H3F3B driver mutations de ne chondroblastoma and giant cell tumor of bone (50). Absence of H3F3A mutation in a subset of malignant giant cell tumor of bone (51). H3F3A promotes lung cancer cell migration through intronic regulation (52). CUL1 is an essential component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, and promotes breast cancer metastasis through regulating EZH2 (53). F-box proteins have pivotal roles in multiple cellular processes through ubiquitylation and subsequent degradation of target proteins (54).
Ultimately, our results suggest that miR-26a-1 affects the expression of cyclin and protein kinase dependent on DNA damage repair in liver cancer stem cells. CDK2 positively regulates aerobic glycolysis by suppressing SIRT5 in gastric cancer (55). MAP kinase dependent cyclinE/CDK2 activity promotes DNA replication (56,57). p21CIP1 promotes cancer-initiating cells via activation of Wnt/TCF1/CyclinD1 signaling (58). CDK8 promotes angiogenesis in pancreatic cancer via activation of the CDK8-β-catenin-KLF2 signaling axis (59). Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2 + extruder at the renal distal convoluted tubule (60). PA28α/β promotes breast cancer cell invasion and metastasis via down-regulation of CDK15 (61). Retinoblastoma protein (pRB) pathway plays a signi cant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis (62). Proliferating cell nuclear antigen (PCNA) is known as a molecular marker for proliferation (63). methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas (64). Phosphoglycerate kinase 1 (PGK1) is an important enzyme in the metabolic glycolysis pathway and the acetylation of PGK1 promotes tumorigenesis (65). p18 blocks reprogramming by targeting Cdk4/6-mediated cell cycle regulation (66).
In conclusions, miR-26a-1 inhibits the expression of NAD+-dependent deacetylase Sirt1 and KDM5A, thereafter, miR-26a-1 promotes DNA damage repair, thereby affecting the expression of some cyclins and protein kinases in liver cancer stem cells. Moreover, excessive Sirt1 and KDM5A abolished miR-26a-1's ability to inhibit the growth of liver cancer stem cells. miR26a-1 may acts as the potential biomarker and therapeutic target for liver cancer. We also shed light on the fact that the attenuation of deregulated functioning of miRNA could be a viable approach for cancer treatment.
Conclusions miR-26a-1 inhibits the expression of NAD+-dependent deacetylase Sirt1 and KDM5A, thereafter, miR-26a-1 promotes DNA damage repair, thereby affecting the expression of some cyclins and protein kinases in liver cancer stem cells. Moreover, excessive Sirt1 and KDM5A abolished miR-26a-1's ability to inhibit the growth of liver cancer stem cells. miR26a-1 may acts as the potential biomarker and therapeutic target for liver cancer. We also shed light on the fact that the attenuation of deregulated functioning of miRNA could be a viable approach for cancer treatment. All methods were carried out in "accordance" with the approved guidelines. All experimental protocols "were approved by" a Tongji university institutional committee. Informed consent was obtained from all subjects. The study was reviewed and approved by the China national institutional animal care and use committee.

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Availability of data and material 'Not applicable' Competing interests "The authors declare that they have no competing interests" Authors' contributions Dongdong Lu conceived the study and participated in the study design, performance, coordination and manuscript writing. Liyan Wang, Xiaonan Li, Rushi Qin, Yanan Lu, Shuting Song, Yingjie Chen, Sijie Xie, Xiaoxue Jiang performed the research. All authors have read and approved the nal manuscript.