SRSF3 Regulates The Sensitivity Of Oral Squamous Cell Carcinoma Cells To Paclitaxel And Cooperates With SRSF9 To Regulate The Alternative Splicing Of PKM

Part I Downregulation of SRSF3 sensitizes oral squamous cell carcinoma and breast cancer cells to paclitaxel treatmentObjectiveOncogene SRSF3(Serine and arginine splicing factor 3)plays important role in tumorigenesis.However,the function of SRSF3 in tumor chemotherapy is still unclear.Paclitaxel(PTX)is widely used in the chemotherapy of many cancers,including breast cancer and oral squamous cell carcinoma(OSCC),but many patients respond poorly to PTX treatment.This study aimed to understand the functions of SRSF3 in PTX treatment and improve the therapeutic effects of PTX treatment.Materials and Methods1.OSCC cell line CAL 27 was treated with different concentrations of PTX(0,7.8n M and 15.6n M).RT-PCR was used to screen for the splicing factors whose m RNA expression was altered by paclitaxel treatment.2.RT-PCR was used to analyze the alternative splicing of SRSF3 exon 4 in breast cancer tissues we acquired.Online program TSVdb(http://www.tsvdb.com)was used to analyze the alternative splicing of SRSF3 exon 4 in breast cancer tissues in TCGA datasheet.3.Lentivirus recombinant plasmid T7-SRSF3 or vector plasmid pLVX-IRES-puro together with packaging plasmids p MD2.G and ps PAX2 were co-transfected into 293 T cells for lentivirus packaging.OSCC cell lines CAL 27,SCC-9 and breast cancer cell lines MCF-7 were then infected to construct cell lines stably overexpressing SRSF3.To inhibit SRSF3 expression,CAL 27,SCC-9 and MCF-7 were transfected with SRSF3-specific antisense oligonucleotide SR-3.The function of SRSF3 in PTX treatment was analyzed by gain-of-function or loss-of-function assay in OSCC cell lines CAL 27 and SCC-9 and breast cancer cell line MCF-7,respectively.4.Alternative splicing of SRSF3 exon 4 in cancer tissues or cells was analyzed by RT-PCR or q RT-PCR.The relationship between exon 4 and tumor prognosis was analyzed by Graph Pad Prism software.5.The effects of PTX and antisense oligonucleotide SR-3 on cell proliferation were measured by cell counting experiments;cells apoptosis induced by PTX and SR-3 were detected by flow cytometry.6.The expression of SRSF3 full-length protein and truncated protein were detected by Western blot.Results1.In CAL 27,the m RNA level of SRSF3 was significantly downregulated after PTX treatment(p<0.05).This was further confirmed in SCC-9 and MCF-7.The expression levels of SRSF3 protein were also significantly inhibited by PTX in these cell lines.These results indicate that PTX inhibits SRSF3 expression.2.Previous experiment proved that SRSF3 overexpressed in OSCC tissues;Here,we discovered that the transcription levels of SRSF3 in breast cancer tissues were higher than their adjacent normal tissues(P<0.05).We further confirmed that SRSF3 is significantly overexpressed in breast cancer tissue compared with normal controls(P<0.01;N=112,T=1093)in TCGA database.Stably overexpressed T7-SRSF3 can partially rescue cancer cell proliferation inhibited by PTX in both CAL 27 and MCF-7(CAL 27: P<0.001;MCF-7: P<0.001).These results indicate that SRSF3 reduces the sensitivity of tumor cells to PTX treatment.3.SRSF3 is a splicing factor and also regulates the alternative splicing of its own exon 4.We examined the effect of PTX on the alternative splicing of SRSF3 exon 4 and found that PTX treatment significantly increased the ratio of SRSF3 long isoform(exon 4 inclusion)and short isoform(exon 4 skipping).Due to SRSF3 exon 4 contains an in-frame stop codon,the long isoform encodes the truncated protein of SRSF3.The short isoform without exon 4 can encode the full-length protein of SRSF3.Therefore,PTX treatment can significantly reduce the expression of SRSF3 full-length protein.These results indicate that PTX inhibits SRSF3 expression by modulating auto-regulation of SRSF3 splicing.4.In order to analyze the difference of SRSF3 exon 4 alternative splicing between tumor tissues and normal tissues,we analyzed the data of breast cancer and OSCC patients in the TCGA database.The results showed that the exclusion of SRSF3 exon 4 in cancer tissues was significantly higher than normal controls(P<0.01);low ratio of exon 4 exclusion vs inclusion isoform significantly correlates with good overall survival in patients with head and neck cancer(P=0.049)or breast cancer(P=0.004).5.The previous research of our group invented the SRSF3-specific antisense oligonucleotide SR-3.Here,we used SR-3 to increase the sensitivity of cells to paclitaxel treatment.In MCF-7 cells,low PTX concentration(0.5 μM)in PTX + NS group could not inhibit cell proliferation.However,combined treatment with both 0.5 μM PTX and 20 n M SR-3 significantly inhibited MCF-7 cell proliferation(P<0.05).In CAL 27 and SCC-9 cells,the PTX + SR-3 group also showed significantly less cell proliferation than the PTX + NS group(CAL 27: P<0.01;SCC-9: P<0.05).The PTX + SR-3 group showed the lowest SRSF3 protein expression levels and induced more apoptosis cell than PTX or SR-3 treated only.These results indicate that inhibition of SRSF3 by SR-3 can enhance cancer cells sensitivity to PTX.ConclusionSRSF3 reduces the sensitivity of tumor cells to PTX treatment.Downregulation of SRSF3 by ASO sensitizes cancer cells to PTX treatment.Part II SRSF3 and SRSF9 regulate the proliferation of HNSCC cells coordinatelyObjective:In Part I,we have demonstrated that inhibiting SRSF3 can increase the sensitivity of cancer cells to PTX.Splicing factors tend to function coordinately in tumorigenic.In Part II,we focused on searching splicing factors that co-express with SRSF3 to further enhance anti-tumor effects against SRSF3.Methods:1.The online website TSVdb(http://www.tsvdb.com)collected the expression levels of SRSF3 and SRSF9(Serine and arginine splicing factor 9)of tumor patients in TCGA database.The correlation between SRSF3 and SRSF9 expression in tumor tissues collected in TCGA database were analyzed by Spearman correlation.The differences between the expression of SRSF9 in cancer tissues and normal tissues in the TCGA database and the differences between SRSF9 expression levels and clinical indicators of HNSCC were analyzed by Mann-Whitney test.2.The effects of SRSF9 on cancer cell proliferation,clone formation,cell cycle and apoptosis were analyzed by si RNA-mediated knockdown of SRSF9 in HNSCC cell lines CAL 27 and SCC-9 as well as breast cancer cell line MCF-7.RT-PCR and western blot were used to detect the effect of si RNA transfection.3.The expressions of SRSF3 and SRSF9 were inhibited separately or simultaneously in CAL 27.The synergistic effects of SRSF3 and SRSF9 on tumor cell proliferation were analyzed by cell counting.Results:1.Splicing factors often function synergistically in tumors.In the TCGA database,we found that SRSF9 expression was positively correlated with SRSF3 expression significantly(P <0.001)in patients with 8 types of cancer including HNSCC and breast cancer.2.Then,we investigated the expression of SRSF9 in the TCGA database and found that the expression of SRSF9 in HNSCC(P <0.001)and breast cancer(P <0.001)were significantly higher than normal tissues.In HNSCC,higher level of SRSF9 expression was associated with poorer prognosis(P = 0.013),higher pathological grades(P <0.05),and clinical stages(P <0.05).3.Next we examined the role of SRSF9 in OSCC.We found that knockdown of SRSF9 repressed cancer cell proliferation and clone formation,and induced cell apoptosis and increased cell percentage in G2/M phase significantly.These results indicate that SRSF3 can promote OSCC progression.4.Last,we examined the synergistic effect of SRSF3 and SRSF9.We found that inhibiting SRSF3 or/and SRSF9 in CAL 27 can inhibit the growth of CAL 27.Moreover,inhibiting SRSF3 and SRSF9 simultaneously can inhibit cell proliferation to the maximum extent(P <0.05).Conclusion:SRSF9 co-expresses with SRSF3 in cancers;simultaneously inhibiting the expression of SRSF3 and SRSF9 can most significantly inhibit OSCC cell proliferation.Part III SRSF9 and SRSF3 regulate the alternative splicing of PKM in Head and Neck Squamous Cell Carcinoma and Breast CancerObjective:SRSF3 regulates alternative splicing of PKM.PKM generates two subtypes through alternative splicing of two mutually exclusive exons: PKM1 contains exon 9 and PKM2 contains exon 10.The expression of PKM2 increases during tumorigenesis.In the Part II,we verified that SRSF9 co-expresses with SRSF3;simultaneously inhibiting the expression of SRSF3 and SRSF9 can most significantly inhibit tumor cell proliferation.So we speculated that SRSF9 may regulate PKM alternative splicing and promote the expression of PKM2.Therefore,the purpose of Part III is to verify whether SRSF9 can regulate PKM alternative splicing.Methods:1.The correlations between the expression of splicing factors and the ratio of PKM2/PKM1 in TCGA database were analyzed by Graphpad using Spearman correlation.The ratios of PKM2/PKM1 in tumor tissues and adjacent normal tissues in TCGA were analyzed using Mann-Whitney test.2.Immunohistochemical staining was used to analyze the expression of PKM1 and PKM2 in tissue microarray.3.SRSF9 was inhibited by si RNA transfection in HNSCC cell lines CAL 27 and SCC-9 and breast cancer cell line MCF-7.The alternative splicing of PKM exon 9 and 10 was analyzed by RT-PCR and western blot.The glycolytic activity was determined by ATP and lactate assay.4.In CAL 27,the expressions of SRSF3 and SRSF9 were inhibited separately or simultaneously to test whether SRSF3 and SRSF9 can regulate PKM alternative splicing coordinately.Results:1.The analysis of TCGA database showed that the ratios of PKM2/PKM1 in 14 cancers including HNSCC(P <0.001)and breast cancer(P <0.001)were significantly higher than those in normal tissues.Tissue microarray immunohistochemical staining showed that the ratios of PKM2/PKM1 gradually increased from normal epithelium(195.7±70.85,mean±ste),dysplasia tissue(662.4±397.3,mean±se),to OSCC tissue(3466±1837,mean±se).The average of PKM2/PKM1 ratio in OSCC was 17.71 times more than the average ratio in normal epithelium(P <0.01).These results indicate that,during tumorigenesis,the increase of PKM2 expression is significantly higher than the alteration of PKM1 expression.2.TCGA database was applied to further screen splicing factors that may regulate PKM alternative splicing.Three splicing factors including SRSF9,hn RNPF and PTBP1 were positively correlated with the ratio of PKM2/PKM1 both in HNSCC and breast cancer significantly.Among them,PTBP1 has been proved to increase M2 expression in previous publications.3.Knockdown of SRSF9 in cancer cells significantly decreased the ratio of PKM2/PKM1,as well as the generation of lactate and ATP in cancer cells.4.When SRSF3 and SRSF9 were inhibited separately or simultaneously in CAL 27,the expression of PKM2 decreased and the expression of PKM1 increased;the ratio of PKM2/PKM1 decreased the most when both SRSF3 and SRSF9 were suppressed(P<0.05).Cells with inhibition of both SRSF3 and SRSF9 produced the least lactate(P<0.001)and ATP(P<0.05)compared with either treated only.These results proved that SRSF3 and SRSF9 regulate PKM alternative splicing synergistically.Conclusion:SRSF9 and SRSF3 promote PKM2 expression via regulating alternative splicing of PKM coordinately.