Inhibition of Rac1 Attenuates Radiation-Induced Lung Injury while Sensitizes Lung Tumor to Radiotherapy

Backgrounds: There is still little progress in the effective treatment of radiation-induced lung injury (RILI), a key dose-limiting factor for thoracic radiotherapy. Ras-related C3 botulinum toxin substrate1, Rac1, is a small guanosine triphosphatase involved in various mechanisms of radiation-induced damage and is over-expressed/mutated in various tumors. The gain-of-function mutation of Rac1 mediates tumor cells’ resistance to radiotherapy. Therefore, inhibiting Rac1 has the potential of protecting normal tissues from radiation-induced injury, and at the same time, sensitizing tumor to radiation therapy, which makes it a promising ideal target for radiation protection. To investigate the protective effects and mechanisms of Rac1 inhibition on RILI, and explore the possible mechanisms that mediate the differential effects of Rac1 inhibition on normal lung tissue and tumor cells. Methods: 60 Co radioactive source was used for ionizing radiation (IR). RILI mouse model was constructed. Inuence of Rac1 inhibition which was achieved by Rac1-specic inhibitor, NSC23766, on RILI were studied by H & E and Masson staining, and immunohistochemical staining of vimentin, TGF-βand γ-H 2 AX. Normal mouse lung epithelial cell line, MLE-12, and mouse lung cancer cell line, LLC, were used to study the effects of Rac1 inhibition on the cellular level. RNA-seq analysis was used for screening differential gene expression caused by Rac1 knockdown. The molecular mechanisms of Rac1 inhibition were studied at the cellular level. Subcutaneous tumor-bearing nude mouse model and orthotopic lung tumor-bearing mouse model were constructed to verify the bidirectional effects of Rac1 inhibition in vivo. Results: RILI of mouse was alleviated by intraperitoneal injection of NSC23766. Rac1 inhibition/knockdown reduced the radiation-induced damage of MLE-12 while aggravated that of LLC. Rac1 translocated from cytoplasm to nucleus after radiation. Tumor protein p53-inducible nuclear protein 1, Trp53inp1, was down-regulated by Rac1 knockdown. In vivo (E and F) qPCR analysis of relative gene expression level of Rac1 and Trp53inp1 between Rac1-SH and Rac1-NC MLE-12. Trp53inp1 over-expression (OE) and negative control (NC) MLE-12 cell lines were constructed on the basis of Rac1-NC and Rac1-SH MLE-12 cells, yielding a total of 4 kinds of genetically modied MLE-12 cells, which were Rac1-NC (cid:0) Trp53inp1-NC (represented as NC-NC), Rac1-SH (cid:0) Trp53inp1-NC (represented as SH-NC), Rac1-NC (cid:0) Trp53inp1-OE (represented as NC-OE) and Rac1-SH-Trp53inp1-OE (represented as SH-OE). All 4 kinds of cells were collected before (0Gy) and at designed time points after 10Gy of irradiation. (G, H) Apoptosis ratio detected by a ow cytometer. (I, J) Apoptosis-related proteins, Bax and Caspase-3 were detected by WB analysis. (K, L) Proteins involved in DNA damage response, p-ATR, p-CHK1 and γ-H2AX were detected by WB analysis. ** and *** represented P <0.01 and 0.001 between the corresponding groups, respectively. The error value was expressed as mean±SEM. Experiments were repeated for three times. (E and F) qPCR analysis of relative gene expression level of Rac1 and Trp53inp1 between Rac1-SH and Rac1-NC MLE-12. Trp53inp1 over-expression (OE) and negative control (NC) MLE-12 cell lines were constructed on the basis of Rac1-NC and Rac1-SH MLE-12 cells, yielding a total of 4 kinds of genetically modied MLE-12 cells, which were Rac1-NC (cid:0) Trp53inp1-NC (represented as NC-NC), Rac1-SH (cid:0) Trp53inp1-NC (represented as SH-NC), Rac1-NC (cid:0) Trp53inp1-OE (represented as NC-OE) and Rac1-SH-Trp53inp1-OE (represented as SH-OE). All 4 kinds of cells were collected before (0Gy) and at designed time points after 10Gy of irradiation. (G, H) Apoptosis ratio detected by a ow cytometer. (I, J) Apoptosis-related proteins, Bax and Caspase-3 were detected by WB analysis. (K, L) Proteins involved in DNA damage response, p-ATR, p-CHK1 and γ-H2AX were detected by WB analysis. ** and *** represented P <0.01 and 0.001 between the corresponding groups, respectively. The error value was expressed as mean±SEM. Experiments were repeated for three times.


RILI was Alleviated by Rac1 Inhibition
Compared with the Naive group, the alveolar septum of the IR group at each time points was signi cantly thickened and the alveolar in ltrated with in ammatory cells ( Figure 1A-B), the proportion of collagen bers increased signi cantly from the 1 st week to the 12 th week ( Figure 1C-D), indicating that pulmonary in ammation and brosis was induced by IR. Intraperitoneal injection of NSC23766, an inhibitor of Rac1 14 , could signi cantly alleviate RILI both in the Low dose (4mg/kg) and the High dose (8mg/kg) groups, manifested as reduction in the thickening of the alveolar septum ( Figure 1A-B) and decrease in collagen ber proportion ( Figure 1C-D). Besides, the IR-induced up-regulation of Vimentin, γ-H 2 AX and TGF-β, which were important indicators of epithelial-mesenchymal transition (EMT) 15 , DNA damage and IR-induced injury, were also inhibited by NSC23766 treatment ( Figure 1E-F, Supplemental material, S1A-D). These results supported that inhibition of Rac1 could produce a protective effect on the lung tissue against RIII in mouse model.
2. Radiation-Induced Injury of Mouse Lung Epithelial Cell Line was Alleviated by Rac1 Inhibition/Knockdown MLE-12 was used for investigation of the effects of Rac1 inhibition in vitro. Cell apoptosis was signi cantly induced, and the expression of apoptosis-related proteins, Cleaved Caspase-3 and Bax, signi cantly up-regulated by 10Gy of radiation (Supplemental material, S2B-E). 100μM NSC23766 pretreatment (dose determined by CCK-8 Test in Supplemental material S3A) could reduce the apoptosis ratio (Supplemental material, S3B-C) and decrease the up-regulation of Cleaved Caspase-3 and Bax at 24hs after radiation (Supplemental material, S3D-E).
For further investigating the roles of Rac1 in radiation-induced injury, Rac1 knockdown (Rac1-SH) and control (Rac1-shNC) MLE-12 cell lines were constructed. After verifying the knockdown e ciency by qPCR and WB (Supplemental material, S3), shRNA1 was used for further experiments. 24hs after 10Gy of radiation, the apoptosis ratio and the expression level of Cleaved Caspase-3 of Rac1-SH MLE-12 were signi cantly lower than those of the control group (Figure 2A-D). Moreover, the expression levels of p-ATR, p-CHK1 and γ-H 2 AX, which were all classic indicators of DNA damage response (DDR), were signi cantly reduced by Rac1 knockdown at 0.5h after 10Gy of radiation ( Figure 2E-F). Collectively, our results indicated that radiation-induced injury of MLE-12 were alleviated by Rac1 inhibition/knockdown.

Identi cation of Trp53inp1 as the Critical Downstream Target of Rac1
To further investigate the downstream target of Rac1, RNA-seq analysis was performed. Quality control results proved that the within-group difference of each group was small and the correlation between the two groups was good (supplemental material, S4A-D). Differential genes screening between groups was based on the signi cant level of P 0.05 and fold change of | 2FC| 0.58. A total of 262 differential genes were caused by Rac1 knockdown, of which 205 genes were down-regulated and 57 up-regulated ( Figure   3A-D). Gene ontology and KEGG pathway analyses showed that these genes are mostly involved in cellular processes of transport and catabolism, and cell growth and death, and are related with human diseases including infectious disease and cancers, and may in uence genetic information processes including replication and repair (supplemental material, S4E-G).
According to the molecular biological functions of differential genes and relative literature research, 14 genes of interest were further analyzed by qPCR. Results showed that Rac1 knockdown cell line was successfully constructed ( Figure 3E), 8 of 14 genes were signi cantly down-regulated or up-regulated and changes were consistent with results from RNA-seq analysis (supplemental material, S5B, C, E-I and Figure 3F), 6 of 14 genes were not signi cantly changed (P 0.05) while their trends of up-regulation or down-regulation were consistent with results from RNA-seq analysis (supplemental material, S5A, D, J-M). qPCR results further validated the reliability of RNA-seq. Among these genes, TRP53INP1 was of most interest, which was signi cantly down-regulated by Rac1 knockdown (P 0.001) ( Figure 3F).
Human tumor protein p53-induced nuclear protein 1 (TP53INP1) is a p53 target gene and encodes the TP53INP1 protein 16 which is mainly expressed in the nucleus 16 and mediates p53-dependent apoptosis and DNA damage 17 . The same gene of mice is named Trp53inp1. According to previous research 16 , various cellular stress stimuli including γ-radiation could induce the expression of TP53INP1 which further stimulates the transcriptional activation of p53-target gene promoters. During γ-radiation induced DNA damage, the mRNA level of TP53INP1 in embryonic broblasts (MEF) of p53 +/+ mice was signi cantly increased, while that of p53 -/mice was not 17 , indicating that DNA damage induced TP53INP1 expression is highly dependent on endogenous p53 17 .
To investigate whether the protective effects of Rac1 inhibition was mediated by down-regulating Trp53inp1, Trp53inp1 overexpression (OE) and negative control (NC) MLE-12 cell lines were constructed on the basis of Rac1-NC and Rac1-SH MLE-12 cells, yielding a total of 4 kinds of genetically modi ed MLE-12 cells. The apoptosis ratio of the four groups were similar before IR treatment and were all signi cantly increased 24hs after 10Gy of radiation ( Figure 3G-H). 24hs after 10Gy of radiation, compared with NC-NC group, the apoptosis ratio of NC-OE group was signi cantly higher (P<0.05), that of the SH-NC group signi cantly lower (P<0.001) ( Figure 3G-H). The apoptosis ratio of SH-OE group was signi cantly higher than the SH-NC group (P<0.001) but still lower than the NC-NC group ( Figure 3G-H).
WB assay demonstrated that the expression levels of Cleaved Caspase-3 and Bax in the four groups were increased after IR (Figure 3 I-J). 24hs after IR, compared with the NC-NC group, the expression levels of Cleaved Caspase-3 and Bax in the SH-NC group were signi cantly lower (both P 0.001), however, that of Cleaved Caspase-3 in the NC-OE group signi cantly higher (P 0.001) and that of Bax was of no signi cant difference ( Figure 3 I-J). The expression levels of Cleaved Caspase-3 and Bax in the SH-OE group were signi cantly higher than those in the SH-NC group (both P 0.001).
Consistent with our previous results, the expression levels of p-ATR, p-Chk1 and γ-H 2 AX in the SH-NC group were all signi cantly lower than those of the NC-NC group at 0.5h after radiation ( Figure 3K-L). The expression level of p-ATR in the NC-OE group was signi cantly higher (P 0.001), the p-CHK1 signi cantly lower (P 0.001) and the γ-H 2 AX of no signi cant difference than those of the NC-NC group ( Figure 3K-L).The expression levels of p-ATR and p-CHK1 in the SH-OE group were signi cantly higher than those of SH-NC group (P 0.001 and 0.05, respectively), the expression level of γ-H 2 AX was higher but lack of statistical signi cance ( Figure 3K-L).
Collectively, these results showed that Rac1 knockdown could reduce the apoptosis and DNA damage of MLE-12 after radiation, and Trp53inp1 overexpression could partially reverse this protective effect of Rac1 knockdown.

Radiation Induces Nuclear-Translocation of Rac1 and subsequently Prolonged the Residence Time of p53 in the Nucleus
According to previous study 18 , when cells faced damage caused by IR, p53 translocated from the cytoplasm into the nucleus and initiated the transcription of Trp53inp1, thereby inducing cell cycle arrest and enhancing p53-mediating apoptosis. Therefore, inhibition of Rac1 may affect the reaction of p53 to radiation stimulation, thereby further down-regulate the expression of Trp53inp1. To verify this hypothesis, the localization of Rac1 and p53 before and after 10Gy of radiation were investigated by confocal microscopy. Results showed that the translocation of Rac1 and p53 from cytoplasm to nucleus started from 30 minutes, peaked at 2hs and subsided at 6hs after radiation ( Figure 4A). Compared with the PBS group, the nuclear-translocation of Rac1 was reduced and subsided earlier (from 2hs after radiation) in the NSC group (treated with 100μM NSC23766) ( Figure 4A), and the residence time of p53 in the nucleus and its expression level were signi cantly reduced ( Figure 4A). Besides, the cytoplasmic protein and nucleoprotein of MLE-12 were separated before (0h) and after 10Gy of radiation. Results showed that the expression level of Rac1 in the nucleoprotein were increased from 30 minutes and peaked at 2hs after IR ( Figure 4B). Further, co-immunoprecipitation analysis showed that Rac1 could bind with p53 ( Figure 4C), and inhibition of Rac1 by NSC23766 reduced the up-regulation of p53 induced by IR ( Figure 4C). These results showed that radiation could induce the nuclear-translocation of Rac1 and p53, and inhibition of Rac1 could block the nuclear-translocation of Rac1 and reduce the residence time of p53 in the nucleus, indicating that nuclear-translocation of Rac1 could prolong the residence time of p53 in the nucleus, thereby promoting Trp53inp1 transcription.

Rac1 knockdown Aggravated the Radiation-Induced Injury of LLC
To date, the main mechanism of action of most radioprotective agents under investigation is to reduce ROS injury. Since oxidative stress injury is one of the main mechanisms of radiation-induced damage, the main problem of these agents is that they also have the potential to protect tumor cells from radiationinduced damage, which is a "side effect" that our investigators do not want. It is important to nd a target that can protect normal cells from radiation damage, and in the meantime, do not affect the killing effect of radiation on tumor. Therefore, we further investigated the effects of Rac1 inhibition/knockdown on the radiation-induced injury of mouse lung cancer cells, LLC.
Successful construction of Rac1-SH and Rac1-shNC LLC were veri ed by WB (supplementary material, S6). 24hs after 10Gy of IR, the apoptosis ratio of Rac1-SH LLC was signi cantly higher than that of Rac1-shNC LLC (P 0.001) ( Figure 5A-B) and the expression levels of Cleaved Caspase-3 and Bax signi cantly higher (both P 0.01) ( Figure 5C-D). Compared with each of their own basal levels (0h), the expression levels of p-ATR, p-CHK1 and γ-H 2 AX were signi cantly increased from 0.5h after 10Gy of radiation ( Figure   5E). The expression levels of p-ATR, p-CHK1 and γ-H 2 AX in the Rac1-SH LLC at several time points after IR were signi cantly higher than those of Rac1-shNC LLC (Figure 5 E). Collectively, our experiments indicated that radiation-induced injury of mouse lung cancer cell line was aggravated by Rac1 knockdown.

In Vivo Investigation of the Differential Effects of Rac1 Inhibition/knockdown
First, subcutaneous tumor-bearing nude mice model was constructed with Rac1-NC/SH LLC. The tumor volumes of Rac1-SH group on the 15 th and 24 th days after tumor-bearing were signi cantly smaller than those of the Rac1-NC group (N=7, P 0.05) ( Figure 6 A-B), indicating that the growth of LLC was signi cantly inhibited by Rac1 knockdown.
Further, orthotopic lung tumor-bearing study was designed. Figure 6C was the illustration of orthotopic lung tumor-bearing and LLR operation. There was no signi cant difference in tumor mass between the two groups at one week after tumor-bearing (P=0.568) ( Figure 6D-E). After one week of PBS or NSC23766 (NSC for short) administration in the 2 nd week, within the same main group (NC group or SH group), the tumor mass of NSC subgroup was signi cantly smaller than that of the PBS subgroup (both P 0.01) ( Figure 6F-G). There was no signi cant difference between the tumor mass of NC+PBS group and that of SH+PBS group, and there was no signi cant difference between the tumor mass of NC+NSC group and that of SH+NSC group, either (P=0.2355 and 0.2160, respectively) ( Figure 6F-G). Radiation intervention was then proceeded. Three weeks after tumor-bearing that was one week after radiation, among the four non-irradiated groups (no IR): (1) The tumor mass of the NC+NSC group, SH+PBS group and SH+NSC group were all signi cantly smaller than that of the NC+PBS group (P 0.05, 0.01 and 0.001, respectively) ( Figure 6H-I).
(2) The tumor mass of the SH+NSC group was signi cantly than that of NC+NSC group (P 0.01) ( Figure 6H-I).
(3) There was no signi cant difference between the tumor mass of the SH+NSC group and that of SH+PBS group (P=0.0515) ( Figure 6H-I). These results showed that tumor growth was signi cantly inhibited by radiation or NSC23766-induced inhibition of Rac1 alone, tumor growth of Rac1-SH LLC was signi cantly slower than that of Rac1-NC LLC. Among the four IR groups, only the tumor mass of SH+NSC group was signi cantly smaller than that of NC+PBS group (P 0.05) ( Figure 6H-I), suggesting that radiation itself produced a strong inhibitory effect on tumor and that Rac1 knockdown/inhibition alone could not exert a more signi cant inhibitory effect at this time point, whereas combination of Rac1 knockdown and inhibition could further inhibit tumor growth and show a synergistic effect. No mice from the four no-IR groups survived by the end of the 4 th week, which was possibly due to organ failure caused by tumor invasion, thus data were only provided for the four IR groups. The tumor mass of NC+NSC and SH+PBS groups were signi cantly smaller than that of NC+PBS group (both P 0.05) ( Figure 6J-K), the difference between those of the SH+NSC group and the NC+PBS group was even more signi cant (P 0.01) ( Figure 6J-K). These results showed that the growth of LLC cells was signi cantly inhibited by Rac1 knockdown and that the combination of Rac1 knockdown and inhibition had a stronger inhibitory effect on tumor growth.
As described above, contralateral non-tumor-bearing lungs were collected for H&E staining on one and two weeks after radiation. Purpose of this part of study was to test whether the radiation-induced injury of contralateral lung could be alleviated by Rac1 inhibition in the same mouse model used for the study of Rac1 knockdown effects on tumor growth. Results showed that: (1) One week after radiation, the thickness of alveolar septum among the four no-IR groups were of no signi cant difference with one another ( Figure 6L-M), and those in the corresponding four IR groups were signi cantly thickened by radiation treatment (Figure 6L-M). (2) One week after radiation, the thickness of alveolar septum in the NC+NSC group and the SH+NSC group were signi cantly smaller than those of the NC+PBS group and the SH+PBS group, respectively (P 0.01 and P 0.001, respectively) ( Figure 6L-M), similar results were seen on two weeks after radiation (both P 0.001) ( Figure 6N-O). These results proved that radiationinduced lung in ammation was alleviated by intraperitoneal injection of Rac1 inhibitor. 7. Possible Mechanisms of the Differential Effects of Rac1 Inhibition/knockdown Many studies 11,12 showed that Rac1 was mutated or abnormally activated in tumors, which promoted tumor phenotype and mediated tumor's resistance to various therapies. Consistent with this, Rac1 in LLC cells was signi cantly over-expressed than that in MLE-12 cells (P 0.05) ( Figure 7A-B). What's more, exon sequencing analysis showed that there were two insertion segments in the RAC1 gene of LLC ( Figure 7C-D). The insertions could contain multiple fragments due to the overlap in the sequencing peaks.
Besides, qPCR and WB results showed that the basal expression level of Trp53inp1 in LLC was signi cantly lower than that in MLE-12 (both P 0.001) ( Figure 7E-H). Therefore, due to the extremely low background expression of Trp53inp1, inhibition of Rac1 in LLC cells may not be able to mediate its protective effects of reducing apoptosis and DNA damage by further down-regulating Trp53inp1.
What's more, 10μM~100μM of NSC23766 treatment did not affect the proliferation ability of MLE-12 (supplemental material, S2A), while same doses of NSC23766 signi cantly inhibited the proliferation ability of LLC (supplemental material, S7A). Consistent with these, the proliferation ability of LLC both before (0Gy) and 24hs after 10Gy of radiation were signi cantly inhibited by Rac1 knockdown ( Figure 7I-J), however, those of the MLE-12 were not signi cantly affected (supplemental material, S7B-C). Thus, we further analyzed changes of cell cycle regulatory proteins (cyclins) in LLC. Results showed that the expression levels of Cyclin D1, CDK1 and Cyclin B1 in Rac1-SH LLC were signi cantly down-regulated compared with those in Rac1-shNC LLC at multiple time points ( Figure 7K-N). According to previous research 19 , the main function of Cyclin D1 is to promote cell proliferation, in addition, Cyclin D1 is overexpressed in many cancers including lung cancer 20 and has been regarded as a proto-oncogene.
Cyclin B1 usually begins to be synthesized in the late G1 phase 21 and reaches a certain level after the cell cycle enters the G2 phase. By binding and activating CDK1, Cyclin B1 promotes the shift from G2 phase to M phase 21 . Also, Cyclin B1 is overexpressed in various cancers including lung cancer 22 . Thus, there are many differences in the expression of cyclins between tumor cells and normal cells, which also mediates the high proliferation and differentiation ability of tumor cells. Our results showed that Rac1 knockdown could exert a relatively general inhibitory effect on various cyclins of tumor cells, thereby signi cantly inhibiting the proliferation activity of tumor cells. This proliferation inhibitory effect was not obvious in normal lung epithelial cells.

Discussion
In addition to traditional supportive treatment and anti-in ammatory treatment with steroid 1 , a variety of radioprotective agents are being developed. However, there is only one type of radioprotective agent, amifostine, approved for clinical use 23 , and the obvious side effects and poor tolerability limit its clinical application 24,25 . Besides, how to protect normal tissues from radiation damage and at the same time ensure the targeted killing of tumor cells by radiotherapy is an important clinical dilemma. There is still an urgent need to nd such a molecular target. Through the study and summary of previous research [26][27][28][29] , Rac1 inhibition was expected to be such a target.
The protective effects of Rac1 inhibition was studied at animal and cellular levels. Results showed that the radiation-induced injury of mouse lung and MLE-12 cells were alleviated by Rac1 inhibition. RNA-seq and qPCR analyses showed that Trp53inp1 was down-regulated by Rac1 knockdown. What's more, the nuclear-translocation of Rac1 and p53 were induced by radiation. Inhibition of Rac1 could block the nuclear-translocation of Rac1 and reduce the residence time of p53 in the nucleus, thereby downregulating the expression of Trp53inp1 and reducing the apoptosis and DNA damage of MLE-12 after γradiation.
Interestingly, unlike MLE-12, Rac1 inhibition/knockdown aggravated the radiation-induced injury of LLC. The reasons by which Rac1 inhibition mediates its differential effects are as followed: First, the Rac1 of normal lung epithelial cells stays normal but is over-expressed and mutated in tumor cells, which may lead to different intracellular biological functions. According to previous studies [11][12][13] , over-activation of Rac1 is associated with tumor phenotype and also mediates tumor tolerance to targeted therapy. Consistent with these, our study showed that Rac1 inhibition sensitized LLC to radiation-induced injury.
Secondly, inhibition of Rac1 down-regulated Trp53inp1 in normal cells, the latter mediates p53-speci c apoptosis and DNA damage pathway. Noticing that the expression of Trp53inp1 in LLC is signi cantly lower than that of MLE-12, therefore, it lacks the effects of Rac1 inhibition mediated by down-regulating Trp53inp1 expression, namely reducing p53-related apoptosis and DNA damage. Last but not least, many cyclins are mutated or up-regulated in most tumor cells, which signi cantly increases their proliferation activity 20,22 . Our study showed that Rac1 inhibition signi cantly reduced the expression of various cyclins in and the proliferation activity of LLC. This proliferation inhibitory effect was not obvious in MLE-12.
Finally, the effects of Rac1 inhibition on normal lung tissues and lung tumor were further veri ed in vivo.
Results from nude mice subcutaneous tumor-bearing showed that the tumor volumes were signi cantly reduced by Rac1 knockdown. Orthotopic lung cancer tumor-bearing study showed that the tumor mass was signi cantly reduced by Rac1 inhibition/knockdown, meanwhile, the radiation-induced injury of nontumor-bearing contralateral lungs was signi cantly alleviated by Rac1 inhibition.

Conclusion
Our study supported that inhibition of Rac1 could not only protect normal lung tissue from radiationinduced injury, but also sensitize lung tumor cells to radiation damage, making it a promising intervention target. The protective effects of Rac1 inhibition was achieved partially by down-regulating Trp53inp1 which is crucially involved in the p53-speci c apoptosis and DNA damage signaling pathways. The overexpression and mutation of Rac1 and the extremely low expression of Trp53inp1 in lung tumor cells may be the fundamental reasons for the differential effects of Rac1 inhibition on normal lung tissue and tumor. This is the rst study to prove this bidirectional effect of Rac1 inhibition on the animal level, and also the rst to discover the interaction of Rac1 with Trp53inp1, indicating a new mechanism for the complex interaction between Rac1-signalling and p53-signalling. Our results may also provide some new angles for the prevention and treatment of RILI caused by chest radiotherapy.

Consent for publication
All authors reached an agreement to publish the study in this journal.

Availability of data and material
All data generated or analyzed during this study were included in this published article and its supplemental material.

Con ict of interests
The authors have no potential con ict of interests.   Figure 1 Radiation-induced lung injury was alleviated by Rac1 inhibition. C57BL/6 were randomly divided into four groups which were Naive (no IR+PBS), IR (IR+PBS), Low-Dose (IR+4mg/kg NSC23766) and High-Dose (IR+8mg/kg NSC23766) (N=20). Rac1 inhibition was achieved by intraperitoneal injection of NSC23766, a speci c Rac1 inhibitor. Intraperitoneal injection of PBS or NSC23766 was given once a day for a consecutive three days. 2 hours after the 3rd injection, all mice except those of the Naive group were anesthetized and immobilized in a radiation-speci c box for 25Gy of local lung irradiation (IR). On the 1st, 3rd, 6th and 12th week after IR, 5 mice from each group were sacri ced and lung tissues were collected.  Radiation-induced lung injury was alleviated by Rac1 inhibition. C57BL/6 were randomly divided into four groups which were Naive (no IR+PBS), IR (IR+PBS), Low-Dose (IR+4mg/kg NSC23766) and High-Dose (IR+8mg/kg NSC23766) (N=20). Rac1 inhibition was achieved by intraperitoneal injection of NSC23766, a speci c Rac1 inhibitor. Intraperitoneal injection of PBS or NSC23766 was given once a day for a consecutive three days. 2 hours after the 3rd injection, all mice except those of the Naive group were Page 20/39 anesthetized and immobilized in a radiation-speci c box for 25Gy of local lung irradiation (IR). On the 1st, 3rd, 6th and 12th week after IR, 5 mice from each group were sacri ced and lung tissues were collected.     signi cant difference between the two groups. *** and ### represented P< 0.001 between the corresponding groups. The error value was expressed as mean±SEM. N=3 for ow cytometer analysis.
WB analysis was repeated for three times.

Figure 6
In vivo veri cation of the differential effects of Rac1 inhibition/knockdown, inhibiting lung tumor while protecting normal lung tissue. (A, B) BALB/cJGpt-Foxn1nu/Gpt mice were randomly divided into two Page 34/39 groups, namely the control group (LLC-Rac1-NC) and the Rac1 knockdown group (LLC-Rac1-SH). After ear labeling, mice were subcutaneously injected with 1×106 of corresponding LLC cells on the right hind leg. The control group were injected with Rac1-NC LLC and the Rac1 knockdown group with Rac1-SH LLC.
The size of each mouse's tumor was measured with a vernier caliper on the 12nd, 15th, 18th, 21st and 24th days after tumor-bearing. Tumor volume (mm3) was calculated as the longest diameter (mm)×the  In vivo veri cation of the differential effects of Rac1 inhibition/knockdown, inhibiting lung tumor while protecting normal lung tissue. (A, B) BALB/cJGpt-Foxn1nu/Gpt mice were randomly divided into two groups, namely the control group (LLC-Rac1-NC) and the Rac1 knockdown group (LLC-Rac1-SH). After ear labeling, mice were subcutaneously injected with 1×106 of corresponding LLC cells on the right hind leg.
The control group were injected with Rac1-NC LLC and the Rac1 knockdown group with Rac1-SH LLC.
The size of each mouse's tumor was measured with a vernier caliper on the 12nd, 15th, 18th, 21st and 24th days after tumor-bearing. Tumor volume (mm3) was calculated as the longest diameter (mm)×the shortest diameter (mm)2. (  10Gy of irradiation. (J) was the statistical analysis of each group's mean uorescence intensity. (K-N) WB analysis of cell cycle regulatory proteins. Rac1-shNC and Rac1-SH LLC cells were collected before (0h) and 2~24hs after 10Gy of irradiation. (L), (M) and (N) were the statistical analysis of the relative gray values of Cyclin D1, CDK1, and Cyclin B1 at each time points. ns represented that there was no statistically signi cant difference between the two groups. *, ** and *** represented P< 0.05, 0.01 and 0.001 between the corresponding groups, respectively. Experiment was repeated for three times.

Figure 8
Summary of study. Ionizing radiation induces the nuclear-translocation of Rac1, the latter then binds with p53 and prolongs the residence time of p53 in the nucleus, thereby promoting the transcription of Trp53inp1 which mediates p53-dependent apoptosis. Inhibition of Rac1 with NSC23766 could reduce the nuclear-translocation of Rac1 induced by irradiation, thereby fasting the release of p53 from the nucleus, at last reducing the apoptosis. However, due to the over-expression and mutation of Rac1, and the extremely low expression of Trp53inp1 in tumor cells, inhibition of Rac1 does not reduce the apoptosis of tumor cells after radiation but can inhibit the proliferation ability of tumor cells.

Figure 8
Summary of study. Ionizing radiation induces the nuclear-translocation of Rac1, the latter then binds with p53 and prolongs the residence time of p53 in the nucleus, thereby promoting the transcription of Trp53inp1 which mediates p53-dependent apoptosis. Inhibition of Rac1 with NSC23766 could reduce the nuclear-translocation of Rac1 induced by irradiation, thereby fasting the release of p53 from the nucleus, at last reducing the apoptosis. However, due to the over-expression and mutation of Rac1, and the extremely low expression of Trp53inp1 in tumor cells, inhibition of Rac1 does not reduce the apoptosis of tumor cells after radiation but can inhibit the proliferation ability of tumor cells.

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