Overexpression of KIAA1199 is an independent prognostic marker in laryngeal squamous cell carcinoma

Patient enrollment and arrange follow up

The research was implemented in the Department of ENT, the Third Xiangya Hospital, Central South University. We collected 10 pairs of fresh specimens and their matched adjacent non-cancerous specimens, which were from patients diagnosed with human laryngeal squamous cell carcinoma by pathological examination in February 2018. A total of 105 patients who had been performed curative resection for LSCC were registered from 2009 to 2014. Patients with recurrence of laryngeal cancer or multiple cancers were excluded. No anticancer therapy was given before surgery. Postoperative pathological examination of patients diagnosed with laryngeal squamous cell cancer. Patient clinical data such as sex, age, pathological differentiation, clinical region, T stage, N stage, clinical stage, operation type, neck lymph dissection, smoking status, and drinking status were collected. To investigate the prognostic value of KIAA1199 in postoperative patients, we examined the overall survival rate (OS) of the LSCC patients. The average arrange follow up cycle was 54 months (5 months extent to 10 years). Prior to the start of the study, we obtained the written informed consent of all patients and the approval of The Institutional Review Board of Third Xiangya Hospital, Central South University in accordance with the Helsinki Declaration Guidelines (No. 2018-S084). All tissue samples were treated and anonymous in accordance with ethical and legal standards. The tumor stage was determined according to the tumor, lymph node, metastasis (TNM) grading of the International Union Against Cancer (UICC, 2002).

RNA extraction and real-time RT-PCR

According to the manufacturer’s protocol, the total RNA was isolated from LSCC and matched adjacent tissues by using TRIzol Reagent (Invitrogen, Waltham, MA, USA). Nanodroplet spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure the concentration and purity of total RNA. According to the manufacturer’s instructions, the total RNA was converted to cDNA using a quantitative PCR (qPCR) reverse transcription kit (TOYOBO Life Science, Shanghai, PR China), fresh tissues were used to synthesize cDNA. Real-time RT-PCR was applied three times using a KOD SYBR qPCR Mix Fluorescent Quantitative PCR kit (TOYOBO Life Science, Shanghai, PR China). PCR and data collection were conducted by using an EP Real-time PCR System (Eppendorf Inc., Hauppauge, NY, USA). For standardization, we used GAPDH as an endogenous control. The primers used in our study were purchased from Sangon Biotech (Shanghai, PR China), and the following primer sequences were used: KIAA1199, F primer 5′-CCAGTAACCTGCGAATGAAGA-3′ and R primer 5′-TGGTCCCAGTGGATGGTGTAG-3′. GAPDH, F primer 5′-TTGGTATCGTGGAAGGACTCA-3′ and R primer 5′-TGTCATCATATTTGGCAGGTT-3′. The reaction conditions were 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and 58 °C for 30 s. The relative expression level was determined by the 2^−ΔΔCt method.

Western blotting analysis

Proteins were extracted from LSCC fresh tissue samples and adjacent non-cancerous fresh tissue samples. The Western blotting analysis was carried out according to our previous article (Li et al., 2012). Primary antibodies were used as follows: polyclonal rabbit anti-KIAA1199 antibody (diluted 1:1,000), anti-GAPDH antibody (diluted 1:5,000), and horseradish peroxidase-conjugated secondary antibody (1:10,000).

Immunohistochemistry

One hundred and five formalin-fixed, paraffin-embedded LSCC tissues were used for the immunohistochemistry (IHC) studies. Briefly, the tissue was sliced continuously into approximately four μm section, paraffin was removed from the sections using a graded alcohol series of 100% and 95% in xylene, rehydrated in 75%, and finally washed with PBS. Subsequently, the antigen was prepared with sodium citrate buffer PBS and incubated in 3% H2O2 deionized water for 15 min to inactivate endogenous peroxidase. The sections were washed three times with PBS, incubated with calf serum to block non-specific antigen for 10 min, incubated with polyclonal rabbit anti-KIAA1199 antibody (1:70) at room temperature for 1 hour, washed with PBS three times, and then incubated with secondary antibody at room temperature for 30 min. Sections were washed with PBS three times, stained with diaminobenzidine (DAB) for 4 min, washed three more times with PBS, restained with hematoxylin for 30 s, washed with flowing water, dried and sealed. Dried sections were observed with an optical microscope. The positive control was gastric cancer tissue confirmed by pathological examination, and the adjacent normal tissues from patients with LSCC were used as the negative control.

The positive expression of KIAA1199 was patchy with aggregates of brown granules in the cytoplasm. Semiquantitative analysis was used to determine the percentage of positive cells under the microscope and score the staining intensity. Two senior pathologists of the Department of Pathology were assigned to read the slides in a double-blinded manner, and 3–5 different fields were randomly selected from each IHC staining section for observation. The staining results were semiquantitatively analyzed in terms of staining intensity and percentage of cells with positive expression. Evaluation of dyeing intensity: range, 0–3; colorless (negative) = 0, weak (pale yellow) = 1, medium (brown–yellow) = 2, strong (tan) = 3. Percentage of stained cells: range, 0–3; percent positive cells <5% = 0, 0.5–10% = 1, 10–50% = 2, ≥50% = 3. The score of the two was multiplied to show the positive grade: 0 is negative (−), ≤3 is low expression, and >3 is higher expression.

Statistical analysis

Our results were interpreted with GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 23.0 software package (SPSS, 112 Y.-H. HAO ET AL. Chicago, IL, USA). Chi-square test was used to analyze the associate with KIAA1199 protein expression and clinicopathological characteristics in LSCC patients. Cox regression analysis estimated the risk of death associated with KIAA1199 protein expression. Kaplan–meier method was used to analyze the total survival curve. Other data were analyzed using Student’s t-test, and ANOVA was conducted to determine the differences in two or more groups. All data are presented as the mean ± SD with P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001).

Clinical data

To understand the clinical features of patients, the detailed data of the patients, such as sex, age, pathological differentiation, clinical region, T stage, N stage, clinical stages, operation type, neck lymph dissection, smoking status and drinking status, were collected from their medical records, and these data are summarized in Table 1 for the 105 patients in this study; 103 (98.1%) were men, and 2 (1.9%) were women, ranging in age from 37 to 82 years. T1–T2 stage was detected in 70 patients (66.6%), N1–N3 stage was detected in 20 patients (19%), and the OS time ranged from 6 to 108 months.

Increased expression of KIAA1199 in human LSCC tissues

To uncover the role of KIAA1199 expression in LSCC, we first detected KIAA1199 protein and mRNA expression in 10 pairs of fresh human LSCC specimens and their matched adjacent noncancerous specimens using Western blotting (Figs. 1A and 1B), IHC (Fig. 2) and RT-PCR (Fig. 1C). As shown in, KIAA1199 protein levels were significantly higher in LSCC tissues (1.838 ± 0.3209 vs. 0.9385 ± 0.1363, P = 0.04) (Fig. 1B) than in adjacent noncancerous tissues. RT-PCR revealed that KIAA1199 mRNA expression was considerably lower in adjacent noncancerous tissues (P < 0.001) than in cancer tissues (Fig. 1C). Then, we compared the expression of KIAA1199 in 105 LSCC tissues and their adjacent noncancerous tissues through IHC. There was weak or negative expression of KIAA1199 in adjacent noncancerous tissue but high expression in the cytoplasm of LSCC tissue cells. The positive staining and negative staining rates in LSCC tissues were 52.4% (55/105, Table 1) and 47.6% (50/105), respectively. Semiquantitative analysis showed that KIAA1199 was significantly increased in LSCC tissues. Representative photographs of the immunostaining are shown in Figs. 3A–3F. RT-PCR results for KIAA1199 mRNA levels were agree with the Western blotting and IHC results, showing that KIAA1199 is increased in LSCC tissues.

KIAA1199 expression is associated with pathologic differentiation, T, N, clinical stage, survival status and survival times of LSCC

In order to further reveal the character of KIAA1199 in LSCC, we evaluated the relationship between its expression and the clinicopathological characteristics of LSCC. As shown in Table 2, upregulation of KIAA1199 expression was associated with some clinicopathological parameters: pathologic differentiation (P = 0.002), T stage (P < 0.001), N stage (P < 0.001), clinical stage (P < 0.001), survival time (P = 0.008) and survival status (P < 0.001). However, KIAA1199 expression was not correlated with age, sex, clinical region, smoking status, or drinking status.

Survival assessment: a high level of KIAA1199 is predictive of poor prognosis in LSCC patients

The survival curve was plotted by kaplan–meier method, and the survival time was tested by log-rank test. The results showed that LSCC patients with high KIAA1199 expression had a lower prognosis, and low KIAA1199 expression in LSCC patients (P < 0.001, for OS) was related with considerably longer OS compared with high KIAA1199 expression. The median OS for high KIAA1199 expression was 60 ± 4.113 months and that for low KIAA1199 expression was 96 ± 7.928 months (Fig. 3G). Then analyzed independent prognostic factors for survival in patients with LSCC by using univariate and multivariate Cox proportional hazards analysis. The univariate analysis results (Table 3) showed that age (HR =1.032, 95% CI [1.001–1.063]; P = 0.04), pathologic differentiation (HR = 0.643, 95% CI [0.524–0.789]; P < 0.001), T stage (HR = 1.402, 95% CI [1.139–1.724]; P < 0.001), N stage (HR = 1.679, 95% CI [1.148–2.4577]; P = 0.008), clinical stage (HR = 1.445, 95% CI [1.180–1.769]; P < 0.001), operation type (HR = 0.380, 95% CI [0.222–0.650]; P < 0.001) and KIAA1199 expression (HR = 12.165, 95% CI [5.434–27.233]; P < 0.001) were significantly associated with the OS of LSCC patients. Multivariate survival analysis (Table 4) showed that KIAA1199 expression was statistically significant predictor of OS (HR = 27.937, 95% CI [10.600–73.632]; P < 0.0001) and that age (HR = 1.039, 95% CI [1.003–1.077]; P = 0.0354), clinical stage (HR = 0.704, 95% CI [0.581–0.960]; P = 0.023), operation type (HR = 0.285, 95% CI [0.093–0.870]; P = 0.027), T stage (HR = 0.68, 95% CI [0.529–0.874]; P = 0.003) and smoking status (HR = 0.19, 95% CI [0.057–0.630]; P = 0.007) were independent predictive factors for OS.