AlG3 Induces AURKA to Promote Laryngeal Squamous Cell Carcinoma Metastasis

Objectives To investigate the relationship between ALG3 and AURKA, the expression and potential prognostic value of ALG3 in LSCC, and tothenexplorethe impact of ALG3 in tumorigenic effects. Methods Co-immunoprecipitation assay was detected the relationship between ALG3 and AURKA, Rt-PCR and Western blot was detected the expression of related mRNA and proteins.CCK8 assay, plate colony formation assay Cells, wound healing, migration and invasion assays were used to examine the abilityof proliferation, movement, migration and invasion of LSCC cells. Results ALG3 immediately induced AURKA to promote LSCC metastasis. Moreover, ALG3 highly expressed in LSCC tissues and cells and the expression of ALG3 was positively relatedto tumor size,lymphatic metastasis and poor clinical prognosis. Furthermore, knockdown ALG3 in LSCC cells remarkablyrestraincellular proliferation, migration and invasioninvitroand vivo. Conclusion AlG3 induced AURKA to promote LSCC metastasis and ALG3 maybe potential prognostic value for LSCC. AlG3 induces AURKA to promote laryngeal squamous cell carcinoma metastasis


Introduction
Laryngeal squamous cell carcinoma LSCC is one of the most common substyle of Laryngealcarcinoma 1 . It is also the type of Laryngealcarcinoma that has the highest rate of mortality and morbidity 2 .Radiotherapy, chemotherapy and surgery, alone or in combination were applied to LSCC treatment 3 .However, the 5-year overall survival (OS) rate was 63% 4 . LSCC was di cult to be completely cured due to tumor metastasis.However, speci c molecular mechanism of LSCC metastasis remained unclear.
Our previous research found Aurora kinase A(AURKA) revives dormant LSCC to promote metastasis 3,5 .Therefore, illustrating speci c molecular mechanism of AURKA revives dormant LSCC to promote metastasiswasof great concern. The key moleculesmaybe capable to potential prognostic value and potential targets for clinical LSCC treatment.
Alpha-1, 3-mannotransferase (ALG3), located on the chromosomal region 3q27.1,was an oncogene implicated in multiple malignancies, for instance, non-small cell lung cancer 6 , breast cancer 7 , oral squamous cell carcinoma 8 , acute myeloid leukemia 9 , et al. ALG3, related with early N-glycans synthesis, was located in the endoplasmic reticulum and Golgi apparatus 10 . ALG3was conducive to high-mannose type N-glycans, which promoted cancer progression 11 .AURKA was reportedly regulated architecture of the Golgi apparatus 12 .Therefore, we guess weather ALG3 got in touch with AURKA.
In our study, we rst investigated the relationship between ALG3 and AURKA, and then ALG3 expression level was examined in LSCC tissues and cells. Afterwards, the correlations between ALG3 and clinical features and potential prognostic value were analyzed.Furthermore, the expression of ALG3 was regulated in LSCC cells. We found ALG3 got in touch with AURKA. Moreover, ALG3 highly expressed in LSCC tissues and cells and the expression of ALG3 was related to tumor size,lymphatic metastasis and poor clinical prognosis. Furthermore, knockdown ALG3 in LSCC cells remarkablyrestraincellular proliferation, migration and invasion.
Our previous research found AURKA revives dormant LSCC to promote metastasis. Illustrating speci c molecular mechanism of AURKA revives dormant LSCC to promote metastasis was of great concern.
ALG3was an oncogene implicated in multiple malignancies.Therefore, Co-IP assay was applied to explored the relationship between ALG3 and AURKA ( Figure 1A and 1B). The result demonstrated that ALG3 corelated with AURKA. While, the regulatory mechanism between ALG3 and AURKA was ambiguous.Plasmid construction and transfection were applied in down-regulating the expression of ALG3 in TU686 cells (TU686/sh-ALG3), and a inhibitor (VX680 13 ) was applied in down-regulating the expression of AURKAin TU686 cells (TU686/VX680). Western blot was used to test the effect of downregulation. Results of gure1C and 1D were shown that p-ALG3 and p-AURKA levels were downregulated obviously (*P<0.05, **P<0.01).Results of gure1E and 1F were shownthatp-AURKA level was reduced distinctly, while p-ALG3 level was not changed (**P<0.01).These results suggested that ALG3 corelated with AURKA, ALG3 was the upstream molecular of AURKA and ALG3 may directly induce AURKA to promote LSCC metastasis.

Overexpression of ALG3 in LSCC tissues and cells
In order to explore the role of ALG3 in promoting the metastasis of LSCC, the expression of ALG3 was investigated between LSCC tissues and matched adjacent non-tumor tissues. qRT-PCR was applied to assess the expression level of ALG3 in 30 LSCC patients. Results of qRT-PCR was shown that the mRNA level of ALG3 was observably higher in tumor tissues than non-tumor tissues (**P<0.01, Figure 2A and 2B). Furthermore, ALG3mRNA and protein levels were examined in LSCC cells line (Hep2, NH8, TU686, TU212) and cell line (Hela). The results shown that mRNA and protein levels of ALG3 were higher in Hep2, NH8, TU686 and TU212 cells, whereas lower in Hela cells. Those ndingsexhibited that ALG3 was upregulated in LSCC tissues and cells. Nevertheless, the high expression of ALG3 was related with clinicopathological features remained unknown. 3. ALG3 high expression level was related to clinicopathological characteristics and poor survival in LSCC patients Chi-square test was applied to analyze relationship between ALG3 expression level and clinicopathological featuresof 60 LSCC patients was exhibited in Table 1. The results shown that ALG3 high expression levelwas observably related to tumor size (P=0.037), invasion range (P=0.014), lymph node involvement (P=0.032) and TNM stage (P<0.001). But there were no statistically signi cant relationships between ALG3 expression and other clinicopathological characteristics such as age(P=0.223)or gender(P=0.874).60 LSCC patients were followed up by 5 years, which 27patients had died. The mortalitywas 45% (27 of 60). The median survival time of 36 months for patients with strong ALG3 staining, while the median survival time of 49 months for patients with weak staining (P=0.0398).Above observations indicated thatALG3 high expression level was related to clinicopathological characteristics, poor survival and LSCC malignancy.

Discussion
Tumor metastasis accounts for the majority of cancer-related deaths worldwide and the revival of dormant tumor cells may be one of the mechanisms related to metastasis. However, the precise molecular and cellular regulators involved in this transition remain poorly understood 14 .Aurora kinase A (AURKA), the family of serine/threonine kinases, regulated the process of mitosis, which is necessary for cell division processes 15 . AURKA is frequently ampli ed and/or overexpressed in GI malignancies (including esophageal, gastric, and colorectal and head and neck cancers) [16][17][18][19] . Our previous research found Aurora kinase A(AURKA) revives dormant LSCC to promote LSCC metastasis 3,5 .Therefore, illustrating speci c molecular mechanism of AURKA revives dormant LSCC to promote LSCC metastasiswas of great concern. The key moleculesmaybe capable to potential prognostic value and potential targets for clinical LSCC treatment.
ALG3was an oncogene implicated in multiple malignancies.ALG3 plays an important role in mitotic recombination 20 .Furthermore, ALG3 was conducive to high-mannose type N-glycans, which promoted cancer progression 11 . AURKA was reportedly regulated architecture of the Golgi apparatus 12 .Therefore, we hypothesize whether AURKA interacts with ALG3 to promote the metastasis of LSCC.
In our study, we rstinvestigated the relationship between ALG3 and AURKA. We guess if ALG3 is related to AURKAto promote LSCC metastasis.Results of Co-IP assay indicated that ALG3 correlated with AURKA. To further explore the regulatory mechanism between ALG3 and AURKA.Plasmid construction and transfection were applied in down-regulating the expression of ALG3in TU686 cells, and the inhibitor (VX680) was applied in reducing the expression of AURKA in TU686 cells. Western blot was used to explore the effect of knock-down. Results suggested that ALG3 corelated with AURKA, ALG3 was the upstream molecular of AURKA and ALG3 may direct regulation AURKA to promote LSCC metastasis.
And then qRT-PCR and Western Blot were carried out to assess the ALG3 expression level in LSCC tissues and cells. ALG3 was upregulated in LSCC tissues and cells. To further illustrate the correlation between ALG3 and laryngeal cancer, the correlations between ALG3 and clinical features and potential prognostic value were analyzed.
Chi-square test was applied to analyze relationship between ALG3 expression level. The results indicated that ALG3 high expression levelwas observably related totumor size, invasion range, lymph node involvement and TNMstage. 60 LSCC patients were followed up by 5 years, patients with high expression of ALG3 have a high mortality rate.
According to the above ndings, ALG3 may function as an oncogene, causing malignant progression in LSCC.In order to further explore ALG3 plays a crucial role in the occurrence and development of laryngeal cancer. The expression of ALG3 was regulated in LSCC cells. Plasmid construction and transfection were applied in down-regulating the expression of ALG3in TU686 and NH8 cells. Knockdown ALG3 in TU686 and NH8 cellsremarkablyrestraincellular ability of proliferation, migration and invasion in vitro and vivo.
In conclusion, our studies indicated that ALG3 corelated with AURKA, ALG3 may directly reduce AURKA to promote LSCC metastasis. Overexpression of ALG3 in LSCC tissues and cells. ALG3 high expression level was related to clinicopathological characteristics and poor survival in LSCC patients. And then ALG3 promoted LSCC cellular ability of proliferation, migration and invasion. Taken together, our ndings indicate that AlG3 induces AURKA to promote laryngeal squamous cell carcinoma metastasis. And ALG3 represent potential targets for clinical LSCC treatment.

Co-immunoprecipitation assay
Co-IP assay was performed following the manufacturer's instructions (Thermo Scienti c) 3 .

RNA Extraction and Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then was reversetranscribed to cDNA using PrimeScript Reverse Transcriptase system (Promega, Madison, WI, USA). QRT-PCR was performed to quantify IL-6 mRNA level with the SYBR Green PCR core Reagent kit (Applied Biosystems, Foster city, CA, USA). GAPDH was used as the endogenous reference. Data were analyzed by using the comparative Ct method. Speci city of resulting PCR products was con rmed by melting curves. The primers were designed using Primer Express v 2.0 software (Applied Biosystems, Foster City, CA, USA). The primers used in this assay were: ALG3: forward, 5′-CACCTTCTGGGTCATTCACAGG-3′ and reverse, 5′-GTGTCACCCTGCAGTTGGGTATAGT-3′; GAPDH: forward, 5′-CTCCTCCACCTTTGACGCTG-3′ and reverse, 5′-TCCTCTTGTG CTCTTGCTGG-3′.

Western blot analysis
TU686and NH8 cells were treated with the treatment of AURKA inhibitor (VX680)for corresponding time.
CCK8 assay and Plate colony formation assay Cells 96-well plates seeded 2×103 cells in 100 ul of DMEM. CCK8 (10 ul) was added to every well. Cells were incubated for 2 h and OD450 absorbance values were measured. Cells were seeded into 6-well plates at 1×103 and 2×103 cells/well and cultured for 3 weeks with DMEM. Then cells were washed twice with PBS and stained with crystal violet for 30 min. Finally, cell colonies in every well were counted.
In Vitrocell migration and invasion assays 2×10 5 treated cells after overnight starvation were plated in the coated lters in 100 μl of serum-free medium. And 600μl of medium containing 10% FBS was added to the lower chamber. The insert chambers' membrane was coated by Diluted Matrigel (BD Biosciences) for measuring the cells invasion. All cells were counted under a high-power objective (10x) in random elds. For migration assays, the upper chamber membranes were plated on top of uncoated (Matrigel-free) lters.
In vivo metastasis 4-week-old male immunode cient mice maintained by the animal resources facility of the medical school of Shanghai Jiaotong University. Animal care and experiments are performed following the "guidelines for the care and utilization of experimental animals" and "principles for the utilization and care of vertebrates", and are approved by the ethics committee of experimental animals of the Medical College of Shanghai Jiaotong University. The average volume and bodyweight of the four groups of mice were similar. The experimental animals were grouped according to the randomization formula. Researchers were not aware of the group allocation at the different stages of the experiment during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis. Mice was randomly assigned to 5 groups ( ve in each group).Cells were inoculated via tail vein into the mice. After six months, counting pulmonary metastatic nodules by H&E staining.

Statistical analysis
Statistical analyses were performed using SPSS 13.0 software. The relationship between the ERp19 expression level and clinicopathologic parameters were calculated with the Pearson χ2 test. Signi cant differences between groups were determined using the student t test. Survival data analysis was performed using the Kaplan-Meier and log-rank tests (GraphPad Prism software v6.0).

Declarations Approval and consent
This study was approved by Institutional Ethnic Committee.

Consent for publication
All authors are consent for publication.

Availability of data and material
The data and material during the current study were available from the corresponding author on reasonable request.

Competing interests
There is on competing interests.     Kaplan-Meier survival curves in LSCC on the basis of ALG3 staining. Patients with ALG3 weak staining had a signi cantly benign prognosis than those with strong staining, P=0.0398.