Xi Zhang,Guangyan Lei,Xinwei Zhang

Abstract Objective:To study the effect of Musashi2 gene on the stemness of CD44v6(+) lung cancer stem cells and explore the mechanism.Methods:Fifty clinical lung squamous cell carcinoma tissues and adjacent tissues were collected.The expression of Musashi2 and CD44v6 was detected by qRTPCR.CD44v6(+)and CD44v6(-)SK-MES-1 cells were sorted using the microbead sorting method.CD44v6(-) cells were divided into pcDNA-Musashi2 group and pcDNA-NC group;CD44v6(+) cells were divided into control group and Notch1 inhibitor RO4929097 group.CD44v6(-) cells transfected with pcDNA-Musashi2 were divided into pcDNA-Musashi2 group and pcDNA-Musashi2+RO4929097 group.The sphere formation experiment was used to detect the self-renewal ability of the cells in each group.Western blotting was performed to detect the protein expressions of stem cell markers Nanog,Oct4,Sox2,Notch1,Hey1,and Musashi2.Results:The expressions of Musashi2 and CD44v6 in lung squamous cell carcinoma tissues were higher than those in adjacent tissues (P＜0.05).Compared with CD44v6(-) cells,the spheroidization ability of CD44v6(+) cells was enhanced (P＜0.05),and the expressions of Musashi2,Nanog,Oct4,Sox2,Notch1 and Hey1 were upregulated (all P＜0.05).Compared with pcDNA-NC,the pcDNA-Musashi2 group had enhanced spheroidizing ability (P＜0.05),and the expressions of Musashi2,Nanog,Oct4,Sox2,Notch1 and Hey1 were up-regulated (P＜0.05).Compared with the control group,the spheroidization ability of RO4929097 group was reduced(P＜0.05),and the expressions of Musashi2,Nanog,Oct4,Sox2,Notch1 and Hey1 were down-regulated (P＜0.05),Compared with the pcDNAMusashi2 group,the pcDNA-Musashi2+RO4929097 group had lower spheroidizing ability (P＜0.05),and the expressions of Musashi2,Nanog,Oct4,Sox2,Notch1 and Hey1 were down-regulated (P＜0.05).Conclusion:Musashi2 maintains the stemness of CD44v6(+) lung cancer stem cells through Notch1 signaling.

Keywords Musashi2;CD44v6;Notch1 signaling pathway;cancer stem cells;lung squamous cell carcinoma

Squamous cell carcinoma (SCC) is one of the most predominant types of non-small cell lung cancer(NSCLC).The mortality rate of lung cancer in China is increasing year by year,with more than 500,000 deaths due to lung cancer each year[1].The high malignancy and poor prognosis of SCC in NSCLC require an in-depth study of the mechanisms of SCC development.With the development of oncology research,there was increasing evidence that lung cancer stem cells had the function of regulating lung cancer tumor initiation,self-renewal,metastasis and chemoresistance,and contributed to recurrence,metastasis and radiotherapy resistance in lung SCC [2].However,the mechanism of how lung cancer stem cells promote lung SCC progression remains to be elucidated.Enhanced understanding of the molecular mechanisms of lung cancer stem cells in cancer progression may facilitate the development of effective stem celltargeted therapies and improve the prognosis of patients with lung SCC.

CD44v6 is a variant exon of the hyaluronan receptor CD44,which was first reported to be of diagnostic importance in tumors in 1992 [3].Moreover,in recent years,CD44v6 has received a lot of attention from scholars in the study of various tumors.Studies have shown that CD44v6 was barely expressed in normal tissues but highly expressed in a variety of cancers,including lung cancer,hepatocellular carcinoma,endometrial cancer and ovarian cancer [3-6].Studies have shown that transfection of benign tumor cells using CD44v6 resulted in enhanced malignant features of benign cells,such as invasive ability and metastatic capacity [4].However,few studies have investigated the expression of CD44v6 in lung SCC and the stem cell function and molecular mechanisms of CD44v6(+)lung squamous carcinoma cells.

Musashi2,a member of the Musashi RNA-binding protein family,is a regulator of asymmetric division in Drosophila and has been shown to be associated with stem and progenitor cell functions [7].Musashi2 was expressed in tumors of the hematopoietic system and played an important role in promoting malignant tumor progression [8],and the Notch1 signaling pathway was one of the key mechanisms for the exercise of its role[9].Musashi2 was also observed in CD44v6(+)-mediated hepatocellular carcinoma stem cells to regulate stemness characteristics and cell metastasis through the Notch 1 signaling pathway [4].Musashi2 also promoted the development of pancreatic cancer and increased drug resistance in pancreatic cancer cells [10-11].However,the contribution of Musashi2 in lung squamous carcinoma cells,especially in CD44v6(+) lung squamous carcinoma stem cells,remains unclear.

In this study,we examined the expression of Musashi2 in human lung SCC tissues and lung squamous cell lines,observed the expression of Musashi2 in CD44v6-positive lung squamous carcinoma cells and the effect of Musashi2 on the self-renewal ability and stemness characteristics of lung squamous carcinoma cells,and explored the role of Notch1 signaling in the stemness characteristics of lung squamous carcinoma cells maintained by Musashi2.This study was conducted to investigate the regulatory role of Notch1 signaling in the stemness characteristics of lung squamous carcinoma cells maintained by Musashi2.This study might provide a new target for the diagnosis and treatment of lung SCC.

Materials and methods

Lung SCC specimens

In this study,50 patients with advanced squamous lung cancer who underwent radical resection from September 2018 to December 2019 in our hospital were selected,including 28 males and 22 females;aged 46-74 (62.35±2.57) years;21 cases were stage III and 29 cases were stage IV according to the international lung cancer TNM classification (8th edition)criteria[12].All specimens were collected immediately after tissue excision and stored at-80 °C.To study the expression of Musashi2 and CD44v6 in human lung SCC tissues,50 surgical lung SCC tissues and paraneoplastic tissues were used for real-time fluorescent quantitative PCR (RT-qPCR) and western blotting.The clinical sample collection and testing experiments in this study were reviewed and approved by our medical ethics committee (ethics number:SPCH1AF2019LSL-059).

Reagents and consumables

The human lung epithelial cell line BEAS-2B and human lung squamous carcinoma cell line SK-MES-1 were purchased from Wuhan Procell Biotechnology Co.The cDNA reverse transcription kits PrimeScript RT Master MixSYBR and SYBR Premix Ex TaqTM were purchased from Takara,Japan,and the TRIzol extraction kit was purchased from Shanghai Beyotime Biotechnology Co.The primary antibodies including Notch1,Hey1,Nanog,Sox2,Oct4,Musashi2,GAPDH and the secondary antibody horseradish peroxidase (HRP) coupled antibodies were purchased from Abcam,U.S.A.RPMI 1640 medium,Dulbecco's modified Eagle medium (DMEM),fetal bovine serum(FBS),DMEM/F12 medium,human recombinant epidermal growth factor (EGF),human recombinant basic fibroblast growth factor (bFGF),B27 supplement,and 1% N-2 supplement were purchased from GIBCO,USA.Penicillin/streptomycin was purchased from Invitrogen,U.S.A.Notch signaling pathway inhibitor RO4929097 was purchased from MedChem-Express,U.S.A.Musashi2 overexpression plasmid(pcDNA-Musashi2) and negative control pcDNA-NC were constructed at Shanghai Genehem Technology Co.EasySep™Human PE Positive Selection Kit and magnets were purchased from STEMCELL Technologies,Canada.PE-coupled CD44v6 antibody was purchased from R&D Systems,USA.1% methylcellulose was purchased from Sigma-Aldrich,USA.Ultralow attachment 24-well plates were purchased from corning,USA.

RT-qPCR

Total RNA was extracted from tissues and cells using the TRIzol RNA Extraction Kit according to the instructions.cDNA was reverse transcribed from the obtained total RNA according to the PrimeScript RT Master Mix kit instructions.qPCR experiments were performed using SYBR Premix Ex TaqTM on a realtime quantitative PCR system (Bio-rad) in strict compliance with the manufacturer's instructions.The mRNA expression levels were normalized by comparing the gene expression with the internal reference gene glyceraldehyde-phosphate dehydrogenase(GAPDH).The relative expression level of each target gene was determined from replicate samples using 2-ΔΔCt(Ct:cycling threshold).Primer sequences were as follows:MSI2 forward:5'-GGTGTTAGCCGAGTATTACCTCTGCA-3';reverse:5'-CATGTACTGAGACTGGCTGATGCGCG-3'.CD44v6 forward:5'-CTGCGTTCGGATCAGTACGCTCGCG-3';reverse:5'-AGATGATGAAGACAGCCCTGAAGTC-3'.GAPDH forward:5'-TGAAGGTCGGTG TGAACGGA-3';reverse:5'-ACAAGCTTCCCATT CTCGGC-3'.

Western blotting

The western blotting experimental procedure referred to the method reported in the literature[13].Pyrolysis of tumor tissue or cells in RIPA lysis buffer.After SDS-PAGE separation of proteins with different sizes,the proteins were transferred to PVDF membrane and blocked in 5 % skimmed milk for 1 h.The membranes were then incubated with primary antibodies Notch1（1∶500）、Hey1（1∶500）、Nanog（1∶600）、Sox2（1∶1,000）、Oct4（1∶600）、Musashi2（1∶800）、GAPDH（1∶5,000）overnight at 4 °C.The transfer membranes were subsequently washed and incubated with HRP-coupled secondary antibodies（1：2,000）.GAPDH protein was the internal reference protein.Protein bands were colorized with the SuperSignal West Pico chemiluminescence kit and imaged by the Odyssey two-color infrared laser imaging system from LICOR.Relative gray scale values were analyzed using Image J software.The experiment was repeated 3 times.

Cell culture

BEAS-2B was cultured in RPMI 1640 medium and the human lung squamous carcinoma cell line SKMES-1 was cultured in DMEM medium.All media were supplemented with 10% FBS and 1% penicillin/streptomycin.Cells were cultured at 37°C in a sterile humidified incubator with 5%CO2.

Magnetic bead cell sorting

CD44v6(+) and CD44v6(-) cell subpopulations were subsequently enriched from the SK-MES-1 cell line by magnetic bead cell sorting and the sorting efficiency was determined.CD44v6(+) cells and CD44v6(-)cells were isolated by the EasySep™Human PE Positive Selection Kit.All steps were performed according to the manufacturer's instructions.Cells were suspended in PBS containing 2% FBS and 1 μM EDTA to a concentration of 2×108cells/mL.Cells were subsequently incubated at room temperature for 15 min after the addition of FcR blocker and PE-coupled CD44v6 antibody,at room temperature for 15 min after the addition of selection mix reagent,and at room temperature for 10 min after the addition of magnetic particles.The tubes were placed into magnets and subsequently harvested for CD44v6(+) and CD44v6(-)cells.

Cell processing and grouping

The harvested CD44v6(+) and CD44v6(-) cells were named as CD44v6(+)group and CD44v6(-)group,respectively.The concentration of both cells was adjusted to 5×104cells/mL.

Treatment of CD44v6(-) cells:CD44v6(-) cells were transfected with 2.5 μg/mL pcDNA-Musashi2 and negative control pcDNA-NC for 24 h.The cells were divided into pcDNA-NC group and pcDNA-Musashi2 group.The transfection effect was detected using western blotting.Cells were treated with 5 μmol/mL of RO4929097,an inhibitor of Notch1 signaling(pcDNA-Musashi2+RO4929097),for 24 h while pcDNAMusashi2 transfected cells,and the control group was the same as the pcDNA-Musashi2 treatment group.

Treatment of CD44v6(+) cells:CD44v6(+) cells were treated with 5 μmol/mL of RO4929097 for 24 h.The control CD44v6(+) cells were not treated,so CD44v6(+)cells were divided into the control and RO4929097 groups.

Sphere formation experiment

Spheroid cell culture:cells of each group were suspended in serum-free DMEM/F12 medium containing 100 IU/mL penicillin,100 μg/mL streptomycin,20 ng/mL human recombinant epidermal growth factor(EGF),10 ng/mL human recombinant basic fibroblast growth factor (bFGF),2% B27 supplement,1% N-2 supplement and 1% methylcellulose,followed by inoculation at a density of 5 × 103cells/mL in ultra-low adherence 24-well plates for 24 h.Spheres larger than 100 μM in diameter were counted by microscopy.The experiment was repeated 3 times independently.

Statistical analysis

The results were analyzed using GraphPad Prism 6.0 statistical software.Comparisons between the two groups were performed using thet-test.Statistical analysis was performed using SPSS 22.0.P＜0.05 was considered a statistically significant difference.

Results

Expression results of Musashi2 and CD44v6 in lung SCC patients

The expression levels of Musashi2 and CD44v6 in lung SCC samples were detected by qPCR and western blot analysis,respectively.The results showed that the mRNA expression of Musashi2 and CD44v6 was significantly upregulated in lung SCC tissues when compared with paraneoplastic tissues,and the differences were statistically significant (Figure 1A and Figure 1B),and the mRNA expression of Musashi2 and CD44v6 showed a positive correlation(r=0.636,P＜0.05).In addition,the protein expression levels of Musashi2 and CD44v6 in lung SCC tissues were also up-regulated when compared with those in paraneoplastic tissues,and the differences were all statistically significant(Figure 1C).

Figure 1.Expression of Musashi2 and CD44v6 in lung SCC tissues.(A)mRNA level of Musashi2.(B)mRNA level of CD44v6(C)Protein levels of Musashi2 and CD44v6.(C)Protein levels of Musashi2 and CD44v6.*P＜0.05 vs.paracancerous tissues.1：paracancerous tissues；2：lung SCC tissues.

CD44v6(+) cells had characteristics of lung squa⁃mous carcinoma stem cells

The expression level of CD44v6 was significantly higher in SK-MES-1 when compared with the BEAS-2B group of lung epithelial cells,and the difference was statistically significant (Figure 2A,P＜0.01).CD44v6(+) and CD44v6(-) cells were isolated from the cell lines in SK-MES-1 by magnetic bead cell sorting.The stemness of CD44v6(+) SK-MES-1 cells was examined in in vitro assays.The results of sphereforming assay showed that CD44v6(+) SK-MES-1 cells could form more spheres than CD44v6(-) SKMES-1 cells (Figure 2B,P＜0.05).Western blotting showed that the expressions of stemness-related genes Nanog,Oct4 and Sox2 proteins were upregulated in the CD44v6(+) group when compared with the CD44v6(-)group,and the differences were all statistically significant(Figure 2C-2F,P＜0.05).

Figure 2.Expression and stemness changes of Musashi2 in CD44v6(+)and CD44v6(-)SK-MES-1 cells.(A)Differences in CD44v6 expression in BEAS-2B and SK-MES-1 cells;**P＜0.01 vs.BEAS-2B.(B) Comparison of the number of sphere-forming cells in CD44v6(+) and CD44v6(-) SK-MES-1 cells.(C) Protein expression bands of stemness-related genes Nanog,Oct4 and Sox2 in CD44v6(+) and CD44v6(-) SK-MES-1 cells.(D-F) Histogram of protein expression levels of Nanog,Oct4 and Sox2;*P＜0.05vs.CD44v6(-).

Musashi2 maintained the stemness characteristics of lung squamous carcinoma cells

Musashi2 expression levels were upregulated in CD44v6(+) cells when compared with CD44v6(-)cells,and the difference was statistically significant(Figure 3A,P＜0.05).Musashi2 was overexpressed by using pcDNA-Musashi2 in CD44v6(-) cells,and the difference was statistically significant with upregulation of Musashi2 in the pcDNA-Musashi2 group when compared to pcDNA-null(Figure 3B,P＜0.05).And the sphere-forming assay analysis showed that the sphere-forming of CD44v6(-)cell was up-regulated in the pcDNA-Musashi2 group when compared with pcDNA-null,and the difference was statistically significant(Figure 3C,P＜0.05).Finally,the pcDNAMusashi2 group up-regulated the protein expression of stemness-related genes (Nanog,Oct4 and Sox2) in CD44v6(-)lung squamous carcinoma cells when compared with pcDNA-null,and the differences were all statistically significant(Figure 3D-G,P＜0.05).

Changes in Notch1 signaling pathway in lung squa⁃mous carcinoma cells

First,the expressions of both Notch1 and Hey1 proteins were down-regulated in CD44v6(-) cells when compared with CD44v6(+) cells,and the differences were all statistically significant (Figure 4A-4C,P＜0.05).Moreover,in CD44v6(-) cells,the expressions of Notch1 and Hey1 proteins were both upregulated in the pcDNA-Musashi2 group when compared with pcDNA-null,and the differences were all statistically significant(Figure 4D-4F,P＜0.05).

Figure 3.Musashi2 maintained the stemness characteristics of lung squamous carcinoma cells.(A) Differential expression of Musashi2 in CD44v6(+) and CD44v6(-) SK-MES-1 cells.*P＜0.05 vs. CD44v6(-).(B) Comparison of the number of sphere-forming CD44v6(-) cells transfected with pcDNA-null and pcDNA-Musashi2.(C-G) Musashi2 and protein expression bands and histograms of stemness-related genes Nanog,Oct4 and Sox2 in CD44v6(-) cells in pcDNA-null and pcDNA-Musashi2 groups;*P＜0.05 vs.pcDNA-null.

Figure 4.Changes in Notch1 signaling pathway in lung squamous carcinoma cells.(A)-(C)Differential expression of Notch1 signaling pathway proteins Notch1 and Hey1 in CD44v6(+)and CD44v6(-)SK-MES-1 cells.*P＜0.05 vs.CD44v6(-).(D-F)Differential expression of Notch1 signaling pathway proteins Notch1 and Hey1 in CD44v6(-) SK-MES-1 cells in pcDNA-null and pcDNAMusashi2 groups;*P＜0.05 vs.pcDNA-null.

Inhibition of Notch1 signaling pathway reduced stemness in CD44v6(+) lung squamous carcinoma cells

The Notch1 signaling pathway inhibitor RO4929097 was used on CD44v6(+) cells.As shown in Figure 5A,the number of sphere-forming cells and the protein expressions of Nanog,Oct4 and Sox2 were downregulated in the RO4929097 group compared with the control group(Figure 5A-5E,P＜0.05).

For CD44v6(-) cells,pcDNA-Musashi2 and RO-4929097 were used in combination.Sphere-forming number and protein expression of Nanog,Oct4 and Sox2 were also down-regulated in the pcDNAMusashi2+RO4929097 group when compared with the pcDNA-Musashi2 group(Figure 5F-5J,P＜0.05).

Figure 5.Inhibition of Notch1 signaling pathway reduces the stemness of CD44v6(+)lung squamous carcinoma cells.(A)Comparison of sphere-forming numbers of CD44v6(+) SK-MES-1 cells in control and RO4929097 groups;(B-E) protein expression bands and histograms of stemness-related genes Nanog,Oct4 and Sox2 in control and RO4929097 groups.*P＜0.05 vs.control.(F)Comparison of the number of sphere-forming CD44v6(-) SK-MES-1 cells in the pcDNA-Musashi2 group and pcDNA-Musashi2+RO4929097 group;(G-J) Protein expression bands and histograms of stemness-related genes Nanog,Oct4 and Sox2 in CD44v6(-)SK-MES-1 cells in pcDNA-Musashi2 group and pcDNA-Musashi2+RO4929097 group;*P＜0.05 vs.pcDNA-Musashi2 group.

Discussion

SCC is a class of malignant tumor types with high incidence,and the screening and study of key genes and signaling pathways in SCC can help to study the mechanisms of the occurrence and development of lung SCC,which can provide a key basis for the selection of therapeutic targets and prognostic molecular markers[14].In this study,we first observed high levels of both mRNA and protein expression of CD44v6 and Musashi2 in lung SCC tissues,and determined that CD44v6 was similarly highly expressed in the lung squamous carcinoma cell line SK-MES-1.After sorting out CD44v6(+) and CD44v6(-) SK-MES-1 cells using the magnetic bead method,we found that CD44v6(+) cells had a distinct stemness profile and increased expression of Musashi2 and Notch1 signaling proteins,but the opposite was true for CD44v6(-)cells.To further investigate the effect of Musashi2 on cell stemness,we transfected Musashi2 overexpression plasmids using pairs of CD44v6(-) cells and observed a significant enhancement of cell stemness characteristics,indicating that Musashi2 upregulated the stemness of CD44v6(-) lung squamous carcinoma cells.However,stemness characteristics were reduced in both CD44v6(+)cells and Musashi2 overexpressing CD44v6(-) cells when the Notch1 signaling pathway was inhibited.Our results suggest that Musashi2 regulates the stemness characteristics of Notch1 signaling pathway-mediated CD44v6(+) lung squamous carcinoma cells.

CD44v6 is a variant of CD44 that is capable of participating in cell-cell and cell-matrix interactions and regulates cell migration,lymphocyte homing,inflammation and tumorigenesis [15].There is growing evidence that CD44v6 is one of the major markers of stem cells in a variety of cancers,including colon and prostate cancers[15].In the present study,we demonstrated that CD44v6(+) lung squamous carcinoma cells had self-renewing stemness properties and might be a marker for lung cancer stem cells.Several studies supported our results.For example,that high expression of CD44v6 occurred in lung cancer and was associated with poor prognosis of lung cancer [16].Consistent with this,we found increased expression of CD44v6 in lung squamous carcinoma tissues and cells.Therefore,the present study suggests that CD44v6 may be a marker of lung SCC tumors and is closely related to cancer stem cells.

In this study,we explored the molecular mechanisms involved in maintaining the stemness of CD44v6(+)lung squamous carcinoma stem cells.We found that elevated Musashi2 expression was closely related to CD44v6 expression in lung SCC tissues,such that Musashi2 was elevated in CD44v6(+) cells when compared to CD44v6(-) cells,suggesting that Musashi2 might be a key regulator in the maintenance of CD44v6(+) lung cancer stemness.Musashi2 has a key role in stem cell function and cell fate determination.First,Musashi2 was expressed in stem and progenitor cells and could regulate differentiation [7],and second,Musashi2 was a key regulator of hematopoietic stem cell self-renewal in myeloid leukemia and myelodysplastic syndromes [17].Recently,Fang et al.showed that Musashi2 promoted self-renewal through LIN28A in hepatocellular carcinoma [18].In the present study,Musashi2 was found to be significantly highly expressed in human squamous lung cancer tumors,and patients with higher Musashi2 expression had been shown to have a relatively poor prognosis [18-19].The evidence of stemness characteristics provided in this study suggested that Musashi2 maintained the stem cell properties of CD44v6(+)lung cancer stem cells,and the spheroid-forming ability of CD44v6(-)lung squamous carcinoma cells and the expression levels of the stemness markers Nanog,Oct4,and Sox2 increased by artificial overexpression of Musashi2,which suggested that overexpression of Musashi2 improved stemness in CD44v6(-) cells.These data suggest that Musashi2 is a key regulator involved in maintaining CD44v6(+) lung cancer stem cell self-renewal and tumorigenesis and may open new directions for the treatment of squamous lung cancer.

The Notch1 signaling pathway is an evolutionarily highly conserved signaling that is activated when receptors interact with ligands to regulate proliferation,self-renewal,differentiation,angiogenesis and migration of tumor stem cells[20-21].There is growing evidence that Notch1 signaling promotes self-renewal and stemness characteristics in hepatocellular carcinoma,glioblastoma,and other tumors[22].Studies have also shown that the Nocth1 pathway was critical in promoting and maintaining cancer stem cells[23].Data from the present study showed that Notch1 signaling protein was activated in CD44v6(+)lung squamous carcinoma cells.inhibition of Notch1 signaling in CD44v6(+) cells significantly attenuated their sphereforming ability and the expression of stemness markers Nanog,Oct4,and Sox2.Moreover,when inhibitors of Notch1 signaling were added to CD44v6(-)lung squamous carcinoma cells with overexpressing Musashi2,the cells showed a similar reduction in stemness characteristics.Thus,we further elucidated the critical role of Notch1 signaling pathway in lung squamous carcinoma stem cells,including CD44v6(+)lung squamous carcinoma stem cells.

In summary,this study showed that Musashi2 was positively correlated with CD44v6 expression in lung squamous carcinoma cells.Musashi2 was highly expressed in CD44v6(+)lung squamous carcinoma stem cells and maintained the stemness characteristics of CD44v6(+) lung squamous carcinoma stem cells.The mechanism might be that Musashi2 maintained the stemness of CD44v6(+) lung squamous carcinoma stem cells by activating the expression of Notch1 signaling.This study provides a new molecular target for the targeting of stem cells in lung squamous carcinoma.

AcknowledgementsThis study was developed by Key Research and Development Program of Shaanxi(No.2019SF-319).