Up-regulation of miR-34c-5p inhibits nasopharyngeal carcinoma cells by mediating NOTCH1

: Objective: To explore the correlation between miR-34c-5p and NOTCH1 in nasopharyngeal carcinoma (NPC). Material and Methods: qPCR was employed to quantify miR-34c-5p and NOTCH1 mRNA in NPC, and Western blot to detect NOTCH1. MiR-34c-5p mimics/inhibitor and NOTCH1 siRNA were constructed to analyze the role of miR-34c-5p/NOTCH1 on the biological function of NPC cells. Results: NPC cells showed lower miR-34c-5p expression and higher NOTCH1 expression than normal cells, and up-regulating miR-34c-5p or inhibiting NOTCH1 could strongly suppress the epithelial-mesenchymal transition (EMT), proliferation, invasion and migration of NPC cells, and induceapoptosis of them. Up-regulating miR-34c-5p could inhibit NOTCH1, and miR-34c-5p was negatively correlated with NOTCH1. Rescue experiment results revealed that NOTCH1 upregulation could counteract the changes of cell process induced by increased miR-34c-5p. Conclusions: MiR-34c-5p inhibits the growth of NPC by down-regulating NOTCH1, so up-regulating miR-34c-5p or down-regulating NOTCH1 may become the potential direction of NPC treatment.


Introduction
Nasopharyngeal carcinoma (NPC) is an epithelial cancer type caused by nasopharyngeal mucosal lesions, which is classified into keratinising squamous cell carcinoma, basaloid squamous cell carcinoma, and non-keratinising squamous cell carcinoma according to histologic features [1]. NPC is one of the most common cancers in south China [2], and its inducements are relatively complicated. Birth history, family history of NPC, relatively short leukocyte telomere length, and Epstein-Barr virus (EBv) are risk factors for NPC [3][4][5]. Gene regulation is an crucial environment factor for the development and progression of NPC, so understanding the molecular mechanism in this process is beneficial to MiR-34c is a 77 bp-long non-coding RNA located on human chromosome 11, belonging to the miR-34 family, which can regulate cell process by binding to target gene sequence fragments. MiR-34c inhibits Bcl2 by pairing to the 3' untranslated region (UTR) of Bcl2 gene, thus down-regulating the viability of laryngeal cancer cells and inducing apoptosis of them [6], and it enhances the cell sensitivity to cisplatin by suppressing the SOX9/-catenin/c-Myc pathway.
Moreover, miR-34c in low level is a risk factor for NPC patients [7]. MiR-34c is related with the development and progression of non-small cell lung carcinomas by regulating genes such as AXL, Cdh1, and Fn1 [8,9], and it can also maintain endoplasmic reticulum homeostasis by down-regulating XBP1, thus regulating the tumor mechanism [10].
NOTCH1 and its dominant signaling pathway are important links in the development of many diseases. For instance, one study by Rice et al. [11] showed that knockout of NOTCH1 could suppress the tumorigenesis effect of prostate cancer cells in rats, and could also down-regulate the metastatic ability of prostate cancer and enhance the sensitivity of the cancer to drugs, and one study by Gan et al, [12] confirmed that NOTCH1 in high level would acceleratethe malignant growth and epithelial-mesenchymal transition (EMT) of tongue cancer and suppress their apoptosis. Furthermore, one study by Li et al. [13] revealed that NOTCH1 prevented DNA damage and cell death through cascade reaction among ATM, CHK2 and p53, and one study by Fender et al. [14] revealed that NOTCH1 pathway promoted the EMT of colon cancer cells by regulating CD44, Slug, and Smad.
There are also previous studies confirming that increased NOTCH1 in NPC takes a part in the development of the NPC tumor [15], and miR-34c may be an inhibitor of NPC [16]. MiR-34c-5p is a mature spliceosome of miR-34c. In this study,it was found through detection of NPC tissue samples that compared with corresponding non-tumor normal tissues, NPC tissues showed down-regulated miR-34c-5p and up-regulated NOTCH1, and it was also predicted through Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200302/882285/bsr-2020-0302.pdf by guest on 27 May 2020 bioinformatics tools that NOTCH1 had sequence sites that can bind to miR-34c. At present, the relationship of miR-34c-5p/NOTCH1 axis in NPC and the regulatory mechanism of them remain unclear. Therefore, under such a situation, this study would try to explore the correlation of miR-34c-5p/NOTCH1 axis with NPC by regulating the expression of the two factors in NPC. Before transfection, the culture medium was replaced with a culture medium without fetal bovine serum, and on the day of transfection, the cells were seeded into a 6-well plate at 110 5 cells/well. The miR-34c-5p mimics, miR-34c-5p inhibitor, NC mimics, NC inhibitor, NOTCH1 siRNA, and NC siRNA vectors were all purchased from Shanghai Sangon Biotech Co., Ltd. The cell lines were transfected with a Lipofectamine 2000 transfection kit (Invitrogen, the United States) in strict accordance with the kit instructions. After 8 hours of transfection, the culture medium was replaced with fresh culture medium at 37℃/5%CO 2 .

qPCR assay
Total RNA was extracted using the Trizol method, and the optical density (OD) of the total RNA at 260-280nm was detected using an ultraviolet spectrophotometer, and the RNA with OD 260 /OD 280 >1.8 was used for next experiment. Reverse transcription and PCR amplification and quantification were conducted to RNA using a FastKing one-step reverse transcription-fluorescence quantitative kit (Tiangen Biotech (Beijing) Co., Ltd., FP314) and ABI PRISM 7000 (Applied Biosystems, the United States). The primers of miR-34c-5p and NOTCH1 mRNA were all designed and synthesized by Shanghai Sangon Biotech Co., Ltd. qPCR was carried out under a reaction system referring to the kit specification.
The system consisted of 50 L total volume containing 1.25 L upstream primer, 1.25 L downstream primer, 1.0 μL probe, 10 pg/g RNA template, 5 μL 50×ROX Reference Dye ROX, and RNase-Free ddH 2 O added to adjust the volume. The reaction process included reverse transcription at 50℃ for 30 min (one cycle) and pre-denaturation at 95℃ for 3 min (one cycle), followed by 40 cycles of denaturation at 95℃ for 15 s, and annealing at 60℃ for 30 s. The results were analyzed using an ABI PRISM 7000 instrument, and the data were normalized using the 2- Ct method, with U6 and GAPDH as internal references.

Western blot
Cell protein extract (1 mL) was adopted to lyse cells, and the cell solution was repeatedly pipetted until the cells were completely lysed. Then the cells were centrifuged at 1.610 4 g for 15 min to take the supernatant, and the protein was separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose (NC) membrane. The membrane was let to stand at room temperature for 1 h (blocked by 5% skim milk -phosphate buffer saline (PBS) solution). Subsequently, the protein to be detected was let to stand with β-actin antibody at 4℃ overnight, and the NC membrane was washed with PBS solution three times, added with goat anti-rabbit secondary antibody (HRP conjugant), and then let to stand for 1 h at room temperature. Finally, the NC membrane was washed with PBS solution, and visualized by electrochemiluminescence (ECL) reagent. The internal reference protein was β-actin, and the relative expression level of the protein to be detected was recorded as the gray value of the band to be detected/the gray value of β-actin protein band.

Transwell assay
Transfected cells were prepared into cell suspension through enzymolysis with trypsin, and seeded into the upper compartment containing 200 L solution with 10% fetal bovine serum +1% DMEM at 210 4 cells/well. The lower compartment was added with 500 L DMEM containing 10% fetal bovine serum.  Photographs about cell migration were acquired under a 200-fold microscope. The cells in 3 randomly selected fields were counted, and the values were averaged, and taken as the number of cells penetrating the membrane. The experiment was repeated three times. Invasion was detected in the same way as above steps after 8% matrix gel was laid on the plate, and the number of cells per well was changed to 510 4 .

MTT assay
Transfected cells were subjected to trypsin enzymolysis, and centrifuged to remove enzyme solution, and then added with a fresh culture medium, and pipetted to prepare cell suspension. The cells were seeded into four 96-well plates at 510 3 cells /100 μL in each well, with 3 wells in each group. One plate was taken out every 24 h, added with 5 mg/ml MTT solution at 10 μL/well, and then cultured continuously for 1 h. Subsequently, the medium was removed, and the OD at 570 nm was measured using an enzyme mark instrument. The experiment was repeated 3 times, and a cell viability-time curve was drawn.
After 48 hours of transfection, the dual luciferase reporter gene (Promega) was used to determine the luciferase intensity in strict accordance with instructions.

Statistical analysis
The above index data were input into SPSS20.0 (Asia Analytics Formerly SPSS China) and GraphPad Prism 6.0 for statistical analysis, and the experiment was repeated three times. Measurement data were expressed as the mean+ standard deviation (mean ± SD), compared between groups using the independent-samples T test, and compared among multiple groups using the Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200302/882285/bsr-2020-0302.pdf by guest on 27 May 2020 one-way anova. Post hoc pairwise comparison was carried out by the LSD-t test, and Pearson correlation analysis was carried out to explore the correlation of miR-34c-5p and NOTCH1 mRNA. All data were analyzed using the two-tailed test, and 95% was used as the confidence interval. p＜0.05 indicates a significant difference.

Down-regulated miR-34c-5p and up-regulated NOTCH1 in NPC
In this study, we collected 74 NPC tissue specimens and 47 corresponding non-tumor normal tissue specimens, and quantified miR-34c-5p and NOTCH1 mRNA in those specimens. Figures 1A and 1B showed that compared with corresponding non-tumor normal tissues, NPC tissues showed decreased miR-34c-5p level and increased NOTCH1 mRNA level. We also determined the levels of miR-34c-5p and NOTCH1 in human nasal epithelial cells (HNEC) and NPC cell lines (SUNE1, CNE2, HK1, and HONE1). Figures 1C and 1D showed that miR-34c-5p was lowly expressed in NPC cells, while NOTCH1 was highly expressed in them. Because miR-34c-5p showed the lowest expression and the second lowest expression in HK1 and CNE2 cells among those cells, respectively, HK1 and CNE2 cells were selected as research objects for this study. We constructed miR-34c-5p mimics and miR-34c-5p inhibitor expression vectors, and transfected them into HK1 and CNE2 cells, and adopted the qPCR and Western blot to determine the level of NOTCH1 in the transfected cells. Figures 1E and 1F showed that up-regulation of miR-34c-5p could inhibit NOTCH1, while down-regulation of it could up-regulate NOTCH1. The above results revealed that miR-34c-5p was lowly expressed in NPC, while NOTCH1 was highly expressed in it, and miR-34c-5p may be involved in regulating NOTCH1. In order to explore the effects of miR-34c-5p on NPC cells, we constructed miR-34c-5p mimics and miR-34c-5p inhibitor expression vectors to regulate miR-34c-5p in HK1 and CNE2 cells. The qPCR results (Figure 2A) revealed that the expression of miR-34c-5p was up-regulated in the miR-34c-5p mimics group, while the expression was down-regulated in the miR-34c-5p inhibitor group. We applied the Western blot assay to determine the expression of pro-apoptotic proteins (Caspase-3, Caspase-9, and Bax) and anti-apoptosis protein (Bcl2). Figure 2B showed that up-regulation of miR-34c-5p gave rise to an increase in the expression of Caspase-3, Caspase-9, and Bax, and a decrease in the Bcl2 expression, while down-regulation of it gave rise to opposite effects. It was found that N-cadherin not only participated in cell invasion together with E-cadherin, but also regulated EMT together with Vinmentin, and -catenin affected cell migration.
The results of Western blot assay ( Figure 2D) revealed that up-regulation of miR-34c-5p promoted E-cadherin, but inhibited N-cadherin, -catenin, and Vimentin, and the results of Transwell assay (Figures 2C and 2F) also revealed that up-regulation of miR-34c-5p inhibited cell invasion and migration. Moreover, the results of MTT assay ( Figure 2E) revealed that up-regulation of miR-34c-5p inhibited cell viability, while down-regulation of it enhanced cell viability. The above results indicated that miR-34c-5p could promote cell apoptosis and inhibit cell proliferation, migration, invasion and EMT.

NOTCH1 inhibits cell apoptosis and promotes cell proliferation, migration, invasion, and EMT.
Because of the abnormal expression of NOTCH1 in NPC and the regulation of NOTCH1 by miR-34c-5p, NOTCH1 siRNA vectors were constructed in this study to explore the role of NOTCH1 in NPC cells. The results are shown in Figure 3. The results showed that inhibition of NOTCH1 resulted in up-regulation of Caspase-3, Caspase-9, Bax, and E-cadherinand down-regulation of Bcl2, N-cadherin, -catenin, and Vimentin, and inhibition of NOTCH1 resulted in a decrease in cell viability, migration, and invasion, which indicated that NOTCH1 inhibited cell

MiR-34c-5p targets NOTCH1
It was found that there were sequence fragments on the 3′ UTR of NOTCH1 that bind to miR-34c-5p according to prediction through the Targetscan database.
Co-transfection of NOTCH1-wt and miR-34c-5p mimics into NPC cells decreased the fluorescence activity of the cells, while other co-transfection combinations did not cause obvious change to the activity ( Figure 4A,B). Pearson correlation analysis revealed that NOTCH1 mRNA was negatively correlated with miR-34c-5p ( Figure 4C). The above results suggested that miR-34c-5p could targetedly inhibit NOTCH1 at the post-transcriptional level.

Discussion
NPC is the cancer with the highest incidence among malignant otolaryngological tumors, which seriously compromises the daily life and health of patients. Studying the regulatory mechanism of NPC cells is helpful to the treatment of NPC. miRNA is one of the important members for regulation of NPC cells, and its regulation on downstream target genes is a crucial mechanism of regulating the NPC cells.In this study, it was found through NPC tissue specimen detection that miR-34c-5p was down-regulated in the specimens, while NOTCH1 was up-regulated in them, so we speculated that the abnormal expression of miR-34c-5p and NOTCH1 may be related to the development of NPC. It was also found that inhibition of miR-34c-5p and overexpression of miR-34c-5p could give rise to up-regulation and down-regulation of NOTCH1, respectively. In addition, it was found that there were sequence fragments on 3' UTR of PTEN that bind to miR-34c-5p according to prediction through the Targetscan database. These results indicated that miR-34c-5p may participate in the regulation of NOTCH1 expression in NPC.
In order to analyze the relationship between miR-34c-5p and NOTCH1 and how this relationship affects the development of NPC, we constructed three vectors, miR-34c-5p mimics, miR-34c-5p inhibitor, and NOTCH1 siRNA, to change the expression of miR-34c-5p and NOTCH1 in NPC. When miR-34c-5p was up-regulated, NOTCH1 was down-regulated and the cell proliferation, migration, invasion, and EMT were weakened. Meantime, the cell apoptosis was intensified.
In contrast, when miR-34c-5p was down-regulated, NOTCH1 was up-regulated and the cell proliferation, migration, invasion, and EMT were strengthened. Meantime, the cell apoptosis was weakened. In addition, down-regulation of NOTCH1 also led to an increase in cell apoptosis and a decrease in cell proliferation, migration, invasion and EMT. Dual luciferase reporter gene assay confirmed that miR-34c-5p could bind to NOTCH1. The above results suggested that miR-34c-5p may inhibit the malignant growth and expansion of NPC cells by down-regulating NOTCH1.
Rescue experiment results revealed that up-regulation of NOTCH1 could counteract apoptosis induced by miR-34c-5p mimics, and promote cell proliferation, migration, invasion and EMT. Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200302/882285/bsr-2020-0302.pdf by guest on 27 May 2020 NOTCH1 protein is an essential part of tumor formation, and NOTCH pathway is a key pathway to regulate cell differentiation and self-renewal of stem cells [17]. NOTCH1 could inhibit downstream E-cadherin by activating Slug in mammary epithelial cells, and finally promote the epithelial cells transform to stromal cells [18], and it can down-regulate MEF2 by activating Snail1 in embryonal rhabdomyosarcoma, thus suppressing cell differentiation and intensifying the characteristics of stem cells in tumor cells [19]. In addition, the NOTCH1/Snail axis up-regulates the metastatic activity of non-small-cell lung carcinoma by inhibiting E-adherin [20]. NOTCH1 also affects cell migration, invasion, and proliferation by regulating proteins such as -catentin, NF-B, p53, HIF-1, MAPK, and cdc2 [21][22][23][24][25]. According to the results in this study, the effect of miR-34c-5p on NPC cells may be realized through a series of cascade reactions. In those cascade reactions, miR-34c-5p changes the activity of the functional protein downstream of NOTCH1 by regulating NOTCH1, and the change of the activity induces corresponding changes of cell biological function. Therefore, miR-34c-5p/NOTCH1 is an effective regulator of NPC cells.
This study has discussed the role of miR-34c-5p/NOTCH1 axis in the pathogenesis of NPC, and it holds that miR-34c-5p can inhibit the malignant expansion of NPC cells caused by high expression of NOTCH1. Although this study has described the mechanism of action of miR-34c-5p/NOTCH1 axis from the molecular level, the clinical value of miR-34c-5p and NOTCH1 still needs further evaluation. Considering the abnormal expression of miR-34c-5p and NOTCH1 in NPC, we will investigate whether the two can be applied to early diagnosis and prognosis evaluation of NPC in the future, and whether regulating miR-34c-5p/NOTCH1 axis is beneficial to the treatment of NPC.What is the reason why miR-34c-5p is down regulated in NPC?Is there an upstream factor of miR-34c-5p that regulates miR-34c-5p expression in NPC?These questions are also worth discussing in future studies.

Conclusion
This study has studied the relationship between miR-34c-5p/NOTCH1 and NPC, and has found a possible mechanism of NPC. In this mechanism (Figure 6), miR-34c-5p inhibits cell proliferation, migration, invasion and EMT and promotes cell apoptosis by targeting NOTCH1. Therefore, up-regulation of miR-34c-5p helps to alleviate the malignant growth and metastasis of NPC.      NOTCH1+miR-34c-5p mimics group showed down-regulated E-cadherin, and up-regulated N-cadherin, -catenin, and Vimentin. C, Compared with the miR-34c-5p mimics group, the NOTCH1+miR-34c-5p mimics group showed enhanced cell viability. D, Compared with the miR-34c-5p mimics group, the NOTCH1+miR-34c-5p mimics group showed intensified cell migration. E, Compared with the miR-34c-5p mimics group, the NOTCH1+miR-34c-5p mimics group showed intensified cell migration. * indicates that in comparison with the miR-34c-5p mimics group, p<0.05, and ** indicates that in comparison with the miR-34c-5p mimics group, p<0.01). Each assay was repeated three times. Figure 6 The mechanism of miR-34c-5p/NOTCH1 axis in regulating NPC. MiR-34c-5p inhibited NOTCH1 mRNA by binding to NOTCH1 mRNA, and finally regulated the proliferation, invasion, migration and EMT of NPC cells. Note: The 2 - Ct standardized method was adopted, with U6 or GAPDH as an internal reference gene of miR-34c-5p and NOTCH1 mRNA.