The genetic variants of LINC-PINT are related to head and neck squamous cell carcinoma susceptibility in Chinese population

Background: Head and neck squamous cell carcinoma (HNSCC) is the most leading incident tumor worldwide. Genetic factors act crucial role in the HNSCC progression. Our study is intent to explore the correlation of LINC-PINT polymorphism with the risk of HNSCC in Chinese population. Methods: The case-control study (including 537 HNSCC cases and 533 controls) was performed to determine the relationship between LINC-PINT polymorphisms and HNSCC susceptibility. Odds ratios (ORs) and 95% condence intervals (CIs) were calculated to determine the associations. Results: The current study indicated that rs157916 and rs7781295 in the LINC-PINT gene have a strong signicant correlation with HNSCC risk (OR 1.32, p = 0.042; OR 1.31, p = 0.016). Stratication analyses showed that rs157916 is related to the increased risk of HNSCC in age ≤ 46 years (OR 1.56, p = 0.029). Rs157916, rs16873842, rs7801029, and rs7781295 exhibited an enhanced risk of HNSCC in men (OR 1.82, p = 0.004; OR 1.61, p = 0.028; OR 1.53, p = 0.047; OR 1.62, p = 0.021). Besides, we found that rs16873842 signicantly increased the risk of Nasopharyngeal SCC (OR 4.04, p = 0.015). Rs157916 (OR 1.39, p = 0.028) and rs7781295 (OR 1.30, p = 0.028) had a high susceptibility to Thyroid SCC. Conclusions: This research indicated that polymorphisms in the LINC-PINT gene are signicantly associated with an increased susceptibility to HNSCC, which suggest that LINC-PINT polymorphisms have a signicant role in prevention and diagnosis of HNSCC. LINC-PINT gene chromosome 7q32.3. LINC-PINT a suppressor and cell behaviors by regulating the expression of target gene in laryngeal carcinomas. that polymorphisms in the LINC-PINT gene have a strong association with HNSCC susceptibility. there no study on the relationship of LINC-PINT genetic variants with the risk of HNSCC. we determine the inuence of LINC-PINT polymorphisms on HNSCC risk. that rs7781295 in the LINC-PINT gene a strong signicant correlation with an increased risk of HNSCC. The two SNPs on the LINC-PINT gene may affect the expression level of LINC-PINT, and then change the expression of target gene, thus changing the risk of HNSCC. However, the exact mechanism is not fully understood and more and deeper functional studies are needed to verify it. Stratied analyses showed that rs157916 is relevant to an enhanced susceptibility of HNSCC ≤ Rs157916, rs16873842, rs7801029, and rs7781295 an increased susceptibility to HNSCC in we that rs16873842 Nasopharyngeal SCC risk. Rs157916 and rs7781295 a susceptibility to Thyroid SCC. Our results suggest that the association of LINC-PINT polymorphisms with the susceptibility to HNSCC may rely on the age, gender, and


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the seventh most common solid malignant tumor worldwide (Ferlay, Soerjomataram et al., 2015). There are over 500,000 new con rmed cases every year (Siegel, Ward et al., 2011). HNSCC includes malignant tumors of the oral cavity, pharynx, thyroid, larynx, and other related parts (Leemans, Braakhuis et al., 2011). Due to the limitation of diagnosis and treatment, most patients with HNSCC are in the middle and late stage when they are diagnosed. So far, the key to improve the curative effect of HNSCC is early detection, early diagnosis, and early treatment. This is also one of the main research directions to improve the survival rate of head and neck tumors in the future (Langer, 2012;Yoshizaki, Ito et al., 2012). Although the speci c pathogenesis of HNSCC is still unclear, previous studies have con rmed that risk factors including smoking, drinking, virus infection, environmental factors, and genetic factors exhibit a vital role in the pathogenesis of HNSCC (Akhtar, Sheikh et al., 2012;Sabir, Baig et al., 2012;Smith, Rubenstein et al., 2012). However, many people are exposed to these risk factors, but a very small number of people will suffer from HNSCC (Sturgis and Wei, 2002). Besides, a study showed that relatives with a family history of HNSCC will increase the risk (Negri, Boffetta et al., 2009). Taken together, these suggest that heredity factors carry out a promotional role in HNSCC progression. Long noncoding RNAs (lncRNAs) are de ned as noncoding RNAs longer than 200 nucleotides. More and more evidence shows that lncRNAs can interact with DNA, RNA and proteins at the transcriptional and posttranscriptional levels, thus regulating protein-coding genes' expression and affecting cell growth, survival, migration, and invasion in human cancer (Zhang, Yin et al., 2017). Besides, growing evidence indicated that lncRNAs contribute to HNSCC progression via regulating biological processes of the tumor cells (Ji, Feng et al., 2020;Jiang, Wu et al., 2020). What's more, genetic variants of lncRNAs such as PTENP1, lncRNA H19, and HOTAIR are associated with the susceptibility of HNSCC (Wu, Liu et al., 2018;Xin, Li et al., 2018;Ghapanchi, Ranjbar et al., 2020).
There is often inactivation of tumor suppressor genes in HNSCC (Arora, Aggarwal et al., 2012;Cadoni, Boccia et al., 2012). To explore the role of tumor suppressor genes in the pathogenesis of HNSCC has become one of the hotspots in the basic research of tumors. At present, with the extensive discovery of polymorphic genetic markers at the molecular level in the whole human genome and the development of the theoretical study of population genetics of related problems, to nd and apply molecular genetic markers in the recombination of genetic transmission of HNSCC and to investigate the molecular genetic mechanism has become a new idea for early detection of HNSCC. Long Intergenic Non-Protein Coding RNA, P53 Induced Transcript (LINC-PINT), a tumor suppressor lncRNA, is transcriptionally regulated by P53. Several previous studies showed that LINC-PINT notably downregulates in many human cancers, such as lung squamous cell carcinomas, breast cancer, and endometrial cancer (Marín-Béjar, Mas et al., 2017). A new research demonstrated that LINC-PINT expression is also downregulated in laryngeal tumors, which act a crucial role in the pathology of laryngeal tumors (Yuan, Xiu et al., 2019). Single nucleotide polymorphism (SNP) in gene can impact the gene's expression and structure, which may lead to the development of human tumors. We speculated that the genetic variants in the LINC-PINT gene have an association with the occurrence of HNSCC. However, there is no report on the association between SNPs in LINC-PINT and the risk of HNSCC.
We performed this study to determine the relationship between LINC-PINT genetic variants and the risk of HNSCC. We rstly obtained four SNPs including rs157916, rs16873842, rs7801029, and rs7781295 in the LINC-PINT gene depending on the 1000 genome with a minor allele frequency (MAF) > 0.05. We further detected the genotyping of all SNPs. Besides, we evaluated the association of SNPs with HNSCC susceptibility. We nally determined the associations strati ed by age, gender, and HNSCC types. Our study will provide some candidate biomarkers for the diagnosis of HNSCC.

Subjects for study
In this current study, we recruited 537 unrelated HNSCC patients and 533 healthy subjects from Gansu Provincial Cancer Hospital and the First A liated Hospital of Xi'an Jiaotong University. Each patient was rstly diagnosed clinically and con rmed as HNSCC by histopathological examination. The controls were selected from healthy people who had been examined in the same hospital.
Each subject must meet the following inclusion criteria: 1) without any cancers, 2) without any family history of tumors, 3) without family history of HNSCC. Each subject was informed of the objective of the study and obtained their informed consent. Basic

SNP selection and genotyping
We obtained four SNPs in LINC-PINT included rs157916, rs16873842, rs7801029, and rs7781295 from the 1000 Genomes Project with MAF > 0.05. Genomic DNA from peripheral blood samples was extracted by using a whole-blood genomic DNA extraction kit.
The purity and concentration of the genomic DNA was detected by a NanoDrop 2000C spectrophotometer. Primers for PCR ampli cation were designed by Agena Bioscience Assay Design Suite software. We further did the SNP genotyping by Agena MassARRAY iPLEX platform. The Agena Bioscience TYPER software 4 was performed to organize and analyze the genotyping data.

Statistical analyses
The normal distribution of all variables was examined by the Kolmogorov-Smirnov test. The age and clinical characteristics of case and control group were compared by t-test. The χ 2 test was used to analyze the gender difference between the case and the control group. The Hardy-Weinberg equilibrium (HWE) of SNPs in the controls was evaluated by a Chi-squared test. The distribution of genotypes and allele from SNPs in cases and controls were detected by the exact test or χ 2 test. The impact of LINC-PINT polymorphisms on HNSCC risk was investigated by computing ORs and 95% CIs under allele, dominant, codominant, recessive, and log-additive models by using logistic regression analysis. What's more, we also detected the associations strati ed by age, gender, and HNSCC types.

Characteristics of study subjects
Our study included in 537 patients with HNSCC and 533 healthy controls. The basic characteristics for each participant in cases and controls were showed in Table 1. The average age was 46.87 ± 15.05 years in the cases and 46.62 ± 13.67 years in the controls. No signi cant variations were observed in gender and age between the cases and the controls (p = 0.950; p = 0.782, respectively). Association between LINC-PINT polymorphisms and HNSCC susceptibility Four SNPs including rs157916, rs16873842, rs7801029, and rs7781295 were successfully genotyped. The basic information of each SNP in the current study was showed in Table 2. Each SNP from the controls followed HWE (p > 0.05). We further evaluate the correlation of LINC-PINT genetic variants with HNSCC susceptibility under ve genetic models with adjustment for gender and age.
Our study indicated that two SNPs (rs157916 and rs7781295) have a strong signi cant correlation with HNSCC risk (   p-values were calculated by unconditional logistic regression analysis with adjustment for age and gender. p < 0.05 indicates statistical signi cance.
Highlighted in bold indicates the signi cant association between SNPs and HNSCC risk.

Strati cation analyses for the relationship between polymorphisms and HNSCC risk
We also detected the correlation between LINC-PINT polymorphisms and HNSCC risk strati ed by age and gender (  p-values were calculated by unconditional logistic regression analysis with adjustment for age and gender. p < 0.05 indicates statistical signi cance.

Highlighted in bold indicates the signi cant association between SNPs and HNSCC risk
We further investigated the in uence of LINC-PINT polymorphisms on Nasopharyngeal SCC and Thyroid SCC risk (

Discussion
In the current study, we rstly detected the association of LINC-PINT polymorphisms with HNSCC susceptibility in Chinese population. Our ndings showed that four SNPs including rs157916, rs16873842, rs7801029, and rs7781295 in LINC-PINT are relevant an enhanced susceptibility to HNSCC. Our study may provide new biomarkers for the prevention of HNSCC in Chinese population.
HNSCC is a complicated and multifactorial malignant tumor, which is caused by alcohol consumption, tobacco smoking, environmental and genetic factors. An increasing study indicated that genetic polymorphisms conduce to HNSCC susceptibility. For instance, Li et al showed that rs1801320 polymorphism in the RAD51gene could signi cantly enhance the susceptibility to head and neck cancer ). Huang's study revealed that interleukin-10 genetic polymorphisms increases head and neck cancer risk (Huang, Song et al., 2017), UGT1A1 genetic polymorphisms were associated with susceptibility to HNSCC (Lacko, Roelofs et al., 2010). Besides, Li found that genetic polymorphisms in NFKB1 are associated with a decreased risk in head and neck cancers . The LINC-PINT gene is located on chromosome 7q32.3. LINC-PINT plays a suppressor and can affect cell behaviors by regulating the expression of target gene in laryngeal carcinomas. We speculated that polymorphisms in the LINC-PINT gene have a strong association with HNSCC susceptibility. However, there is no study focusing on the relationship of LINC-PINT genetic variants with the risk of HNSCC. Thus, we tried to determine the in uence of LINC-PINT polymorphisms on HNSCC risk. We observed that rs157916 and rs7781295 in the LINC-PINT gene have a strong signi cant correlation with an increased risk of HNSCC. The two SNPs on the LINC-PINT gene may affect the expression level of LINC-PINT, and then change the expression of target gene, thus changing the risk of HNSCC. However, the exact mechanism is not fully understood and more and deeper functional studies are needed to verify it. Strati ed analyses showed that rs157916 is relevant to an enhanced susceptibility of HNSCC in age ≤ 46 years. Rs157916, rs16873842, rs7801029, and rs7781295 exhibited an increased susceptibility to HNSCC in men. Besides, we observed that rs16873842 signi cantly enhanced Nasopharyngeal SCC risk. Rs157916 and rs7781295 had a high susceptibility to Thyroid SCC. Our results suggest that the association of LINC-PINT polymorphisms with the susceptibility to HNSCC may rely on the age, gender, and the subtypes of HNSCC.
Our study had some weakness. First, we detected the correlation between LINC-PINT polymorphisms and HNSCC risk, the relationship between LINC-PINT polymorphisms and the expression level of this gene in HNSCC should be tested in future. Second, drinking and smoking are the important risk factors for HNSCC, the in uence of LINC-PINT polymorphisms on HNSCC risk under drinking and smoking subgroups will be tested in next. In spites of its shortcomings, our ndings provide the new perspective for the molecular mechanism of HNSCC occurrence.

Conclusion
In conclusion, our present study indicated that LINC-PINT genetic variants have a strong relationship with the HNSCC risk, which suggest that LINC-PINT may carry out an important role in the pathology of HNSCC.