SF3B4 downregulation restrains lung adenocarcinoma tumorigenesis via 5′ alternative splicing of KAT2A

Aberrant expression of splicing factors, including SF3B4, plays a vital role in lung adenocarcinoma (LUAD). However, the impact of SF3B4 in the progression of LUAD has not been studied well. Here, we demonstrated the effects of SF3B4 in LUAD via apoptosis, proliferation, migration assays, etc. Gene manipulations confirmed the role of SF3B4 via KAT2A. SF3B4 was found to promote LUAD growth. Further studies found that, upon SF3B4 knockdown in LUAD cells, an alternative splice site occurred at the 5′-UTR of KAT2A, which led to the downregulation of KAT2A at both RNA and protein levels. Furthermore, the decrease in KAT2A expression partially reversed the effect of SF3B4 in promoting tumorigenesis. The axis SF3B4/ KAT2A was identified as a significant player in LUAD progression, shedding light on the therapeutic development in LUAD.


Aberrant expression of SF3B4 in LUAD
To search critical splicing factors involved in LUAD, overlapping analysis of 2562 upregulated genes and 134 core splicing factors revealed 15 potential candidates, including RBMBA, LSM8, and SF3B4 (Fig. 1A,B).To explore the effects of SF3B4 in LUAD, we examined the mRNA profile of SF3B4 in LUAD and normal lung tissues from the GTEX and TCGA databases (Fig. 1C).Then, we investigated the expression of SF3B4 protein in LUAD and normal lung tissues from the CPTAC database (Fig. 1D).Notably, the mRNA level of SF3B4 was higher in LUAD tissues and LUAD cells than in normal samples (Fig. 1E,F).These findings were further confirmed by comparing the results from the staining of SF3B4 in human tumor tissues to those in healthy tissues (Fig. 1G).
We performed a Kaplan-Meier analysis to explore whether SF3B4 contributed to LUAD progression.We observed that patients with high levels of SF3B4 had worse overall survival (OS) and poor progression-free survival (PFS) (Fig. 1H,I).Next, correlations of SF3B4 to clinicopathological features were analyzed.Importantly, SF3B4 expression was substantially correlated with the TNM stage (Table 1) in LUAD.

Promoting the role of SF3B4 in LUAD
To explore the role of SF3B4 in LUAD, we transfected siRNA targeting SF3B4 into wild-type LUAD cells.The efficiency of SF3B4 knockdown was validated in transcription and translation levels (Fig. 2A).SF3B4 knockdown significantly suppressed the proliferation of LUAD cells (Fig. 2B,D).Besides, SF3B4 knockdown significantly inhibited the formation of colonies derived from A549 and H1650 cells, respectively (Fig. 2E).Furthermore, we found that the knockdown of SF3B4 decreased the capacity to migrate and invade LUAD cells (Fig. 2F,G).Consistently, the knockdown of SF3B4 inhibited the capacity of LUAD cells in wound healing (Fig. 2H).Futhermore, we showed that SF3B4 knockdown induced apoptosis in LUAD cells in vitro (Fig. 2C, Supplementary Fig. 1).These data suggest that targeting SF3B4 could be an effective treatment for LUAD.

SF3B4 promotes LUAD tumor growth
To examine the effects of SF3B4 in vivo, we constructed the cell lines with a knockdown of SF3B4 and a cell line with a control vector.We subcutaneously inoculated sh-SF3B4 and sh-Ctrl cells in mice.After 3 weeks, we found dramatic decreases in tumor size and weights from the mice-bearing tumors derived from SF3B4 knockdown cells compared to the control (Fig. 3A-C).Besides, we quantified the levels of SF3B4 and Ki-67 with immunohistochemical staining in this nude mouse xenograft model.The level of SF3B4 was reduced in the SF3B4 knockdown group (Fig. 3D).The intensity of Ki-67 was lower in tumors derived from SF3B4 knockdown cells than that derived from control (Fig. 3D).In summary, the results demonstrated a promoting role of SF3B4 on LUAD.

KAT2A is an essential downstream effector of SF3B4
RNA-seq was performed in A549 cells with or without SF3B4 knockdown to explore the pathway through which SF3B4 drove the growth and development of LUAD.The profile of differentially expressed genes (DEGs) was plotted (Fig. 4A,B).We found 324 upregulated and 597 downregulated genes.Gene analysis was performed to depict the biological functions of these DEGs.Interestingly, the enriched biological pathways are involved in regulating the cell cycle and the negative regulation of phase transitions in the mitotic cell cycle (Fig. 4C).Exploring the genes that are down-regulated after SF3B4 knockdown could provide valuable insights.For downregulated genes upon SF3B4 knockdown, the enrichment of biological pathways was DNA double-strand break repair, cell cycle regulation, and DNA replication in downregulated genes (Fig. 4E).Interestingly, 23 genes were identified to have both downregulated expressed genes and were involved in the differential AS events in A549 cells (Fig. 4D).We then performed visual analysis on the identified genes, and rMATs found that the 5′ end of KAT2A had www.nature.com/scientificreports/variable splicing.An A5SS variable splicing event occurred in the annotated region of the shadow.When SF3B4 expression is interfered with si-SF3B4, a long exon is generated at the 5′ variable splicing site of KAT2A.This results in the transcription containing the long exon no longer encoding.This reduces the expression of KAT2A protein.(Fig. 4F).To further explore the role of SF3B4 in regulating KAT2A, the changes in the KAT2A level were confirmed by qRT-PCR and Western blotting upon SF3B4 knockdown in LUAD cells, including A549 and H1650.Both mRNA and protein levels of KAT2A were significantly downregulated upon SF3B4 knockdown (Fig. 4G,H).These results suggest that KAT2A is a target of SF3B4 in LUAD.

SF3B4 leads to AS of KAT2A
We further analyzed the splice variants of KAT2A mRNA with an ensemble genome browser to explore if SF3B4 regulated the levels of KAT2A through AS.We found that a long exon was produced in the unspliced transcript of KAT2A-202, different from the spliced transcript of KAT2A-201 (Fig. 5A).KAT2A-201 mRNA level was increased than that of KAT2A-202 in LUAD cells.Furthermore, we analyzed the levels of KAT2A-201 and KAT2A-202 transcripts from both LUAD tissues and normal controls in TCGA and GTEX databases.We found that the expression of KAT2A-201 transcripts in LUAD was relatively higher than that of KAT2A-202, while KAT2A-202 was relatively higher in normal lung tissue (Fig. 5B).RT-PCR results verified the changes in the levels of KAT2A-201 and KAT2A-202.We observed that upon SF3B4 knockdown, the level of the unspliced transcript variant was significantly increased, and that of the spliced transcript variant was significantly reduced (Fig. 5C,D).Interestingly, RIP-PCR data showed that the mRNA level of KAT2A in SF3B4 precipitates was significantly increased than that in the IgG group (Fig. 5E).This suggests that SF3B4 regulates the KAT2A via A5SS.

Targeting KAT2A in LUAD
To evaluate the role of KAT2A alone in LUAD cells, we transiently silenced KAT2A with siRNA transfection into LUAD cells.The knockdown of KAT2A was confirmed (Fig. 6A,B).The analyses revealed that KAT2A knockdown decreased cell viability and colony formation and increased cell apoptosis (Fig. 6C-F, Supplementary Fig. 2).Transwell analysis results showed that knockdown of KAT2A reduced the invasion and migration capacities of LUAD cells (Fig. 6G-H).Furthermore, rescue experiments were performed to confirm the effect of KAT2A in SF3B4-regulated malignancy of LUAD.We transfected si-NC or KAT2A-targeted siRNA into LUAD cells with SF3B4 overexpression (Fig. 7A,B).We observed that proliferation rate, migration, and invasion capacities in LUAD cells enhanced with SF3B4 overexpression were impaired upon the KAT2A knockdown (Fig. 7C-F).These findings suggest the effects of KAT2A in the SF3B4-modified oncogenesis of LUAD.

Discussion
Emerging evidence has manifested that splicing factor SF3B4 binds to U2 snRNP, which regulates the AS of premature mRNA 15 .SF3B4 is an oncogenic driver in multiple tumor types.However, it is a tumor-suppressor in pancreatic cancer [16][17][18][19] .We showed increased levels of SF3B4 in LUAD tissues and cells.The upregulated level of SF3B4 was associated with a poor prognosis of LUAD.Furthermore, we observed that SF3B4 knockdown suppressed proliferation and mobility and induced apoptosis in LUAD cells.Meanwhile, we proved that the SF3B4 knockdown slowed tumor growth in a mouse model.Our investigation showed that SF3B4 exhibited oncogenic properties in LUAD, in line with other studies 14 .
Understanding the underlying mechanisms of SF3B4 as an oncogene requires further studies.In cervical cancer, Li et al. proposed that the knockdown of SF3B4 decreased the splicing efficiency of SPAG5 via retained www.nature.com/scientificreports/intron, leading to a reduction in SPAG5 expression 20 .In hepatocellular carcinoma, the SF3B4 knockdown induced the inactivation of p27 by skipped exon, alternating the splicing of KLF4 9 .Since the splicing effect of SF3B4 has www.nature.com/scientificreports/not been studied well in LUAD, RNA-seq was performed to screen AS events.Through rMAT analysis and RT-PCR validation, we found that an alternative splice event occurred at the 5 ' end of the KAT2A gene, producing a long exon.The transcript containing the long exon does not encode, resulting in the downregulation of KAT2A after SF3B4 knockdown in LUAD.Therefore, for the first time, we found that SF3B4 regulated LUAD progression via the alternative splicing of KAT2A.The 5′ untranslated region (5′UTR) of a mature mRNA is involved in gene regulation at the post-transcriptional level.Within a given transcriptional unit, AS is one event that induces changes to the 5′UTR sequence 21 , leading to differences in protein variant output.Selecting 5′ splice-sites (5′SS) in AS plays essential roles in gene regulation, including cell proliferation, invasion, and apoptosis [22][23][24] .KAT2A, a member of the N-acetyltransferase www.nature.com/scientificreports/superfamily, was identified as a histone acetyltransferase binding to acetyl-CoA 25,26 .Various studies have indicated that KAT2A is an epigenetic oncogene in several malignancies [27][28][29] .Besides, KAT2A has been reported www.nature.com/scientificreports/ to promote stem-like cell propagation in leukemia and regulate the resistance to tamoxifen in breast cancer 30 .However, little was known about the effects of KAT2A in LUAD.Here, we confirmed that KAT2A knockdown

Figure 1 .
Figure 1.Aberrant expression of SF3B4 in LUAD.(A) Venn diagram showing the 15 genes correlated with upregulated genes in TCGA LUAD (n = 2562) and involved in cone splicing genes (n = 134).(B) Heatmap of selected genes in (A).(C) Relative mRNA expression of SF3B4 in LUAD and normal lung tissues from GTEX and TCGA and database.(D) Expression of SF3B4 protein in LUAD and norma lung tissues from CPTAC database.(E) SF3B4 mRNA levels in A549, H1650, and HPAEpiC cells, measured by qRT-PCR.(F) SF3B4 mRNA level in human lung tissues (n = 36), analyzed by qRT-PCR.(G) IHC staining of SF3B4 in human LUAD tissues and corresponding healthy tissues.(H,I) Kaplan-Meier analysis on the effects of SF3B4 on PFS and OS of LUAD patients.* P < 0.05, ** P < 0.01.

Figure 3 .
Figure 3.Effect of SF3B4 on LUAD growth in vivo.(A) Images of mice with tumors (n = 5).(B) Tumors derived from sh-SF3B4 cells or control cells.(C) Tumor weights and size.(D) IHC staining results for SF3B4 and Ki-67 in tumor tissues from a mouse model.* P < 0.05, ** P < 0.01.

Figure 4 .
Figure 4. KAT2A is a downstream target of SF3B4.(A,B) Heatmap and volcano plots of DEGs from RNA-seq in A549 cells with or without SF3B4 knockdown.(C) GO analysis of DEGs.(D) Venn diagram of 3022 genes related to differential AS events upon knockdown of SF3B4.(E) GO analysis of downregulated DEGs (F) RNAseq reads mapping to KAT2A in A549 cells upon knockdown of SF3B4.(G,H) qRT-PCR and western blotting of KAT2A levels upon SF3B4 knockdown in A549 cells.* P < 0.05, ** P < 0.01.Original blots/gels are presented in Supplementary Raw data.

Figure 6 .
Figure 6.Effects of KAT2A on proliferation and mobility of LUAD.(A,B) qRT-PCR and western blotting on the efficiency of KAT2A knockdown by targeted siRNAs in LUAD.(C) CCK-8 test on cell proliferation.(D) Apoptosis assay on A549 and H1650 cells with or without KAT2A knockdown.(E) EdU test on cell proliferation.(F) Clonogenic assay on A549 and H1650 cells.(G, H) Transwell assays on A549 and H1650 cells.* P < 0.05, ** P < 0.01.Original blots/gels are presented in Supplementary Raw data.

Figure 7 .
Figure 7. Effects of KAT2A on proliferation and mobility of LUAD cells with SF3B4 overexpression.(A,B) qRT-PCR and western blotting on the efficiency of KAT2A knockdown in LUAD cells with or without SF3B4 overexpression.(C) CCK-8 test on cell proliferation.(D) EdU test on cell proliferation of A549 and H1650 cells.(E,F) Transwell assays on A549 and H1650 cells upon KAT2A knockdown, with or without overexpression of SF3B4.* P < 0.05, ** P < 0.01.Original blots/gels are presented in Supplementary Raw data.