Introduction

Stroke includes hemorrhagic stroke and IS1, stroke incidence has risen by approximately 8.7% annually, which is a serious threat to people’s health. In the worldwide, approximately 15 million people suffer from stroke annually, of which IS accounts for about 80%2,3. IS, also known as a cerebrovascular accident, is a disease caused by cerebral ischemia and hypoxia, which can leads to the weakening of brain-innervated nerve function4,5. It has the characteristics of high incidence, high disability rate, high mortality rate, high recurrence rate and high economic burden6,7. At present, the methods of IS clinical diagnosis mainly include CT, cerebral angiography, magnetic resonance imaging and cerebral blood flow measurement8. IS patients have the symptoms of sudden vertigo, diplopia, bilateral limb numbness and weakness, severe patients will have the symptoms of hemiplegia, hemianopsia, aphasia, sensory disorders, consciousness disorders and death9,10. There are a number of risk factors can cause IS, which include smoking, alcohol drinking, hyperlipidemia, diabetes mellitus, genetic factors, atrial fibrillation and genetic mutations11,12. It has been reported that IS risk is closely related to genes13,14, so more and more people have begun to explore the method of prevention and treatment IS from the genetic aspect. In recent years, it has been found that HIF1A is related to the pathological process of a variety of diseases, however, the relationship between HIF1A and IS risk is still unclear, which needs further study. HIF1A, a heterodimeric transcription factor, is the key transcriptional regulator of intracellular adaptive hypoxia environment, it plays a key role in the signal transmission between hypoxia and angiogenesis15,16,17. Relevant studies have shown that HIF1A is involved in a variety of biological functions, such as cell cycle, apoptosis and angiogenesis18,19. When the body is hypoxic/ischemic, HIF1A will accumulate in ischemic and hypoxic tissues, then it will bind with specific target genes, which can regulate vascular endothelial growth factor level and activate the pathological processes of ischemic injury20, so we explored the relationship between HIF1A protein levels and IS.

HIF1A is a hypoxia response core regulator, its gene polymorphisms have been associated with various diseases susceptibility, among which ‌rs10873142, ‌rs11549465 and ‌rs11549467 have become the focus of disease association studies21,22,23,−24. HIF1A rs10873142 polymorphism T allele was previously demonstrated to be associated with a higher transcriptional activity and increased angiogenesis, it is associated with the risk of coronary artery disease25, so it may be associated with the IS risk. HIF1A rs11549465 can regulate the HIF-1 A expression, because it can inhibit ubiquitin-proteasome degradation to cause HIF-1 A protein accumulation, which can increase the expression of target genes, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO), then exacerbate the leakage of blood-brain barrier and the release of inflammatory factors in the ischemic penumbra26. HIF1A ‌rs11549467 is associated with cardiovascular system diseases, because it can inhibit hypoxia response element-mediated activation of glycolysis related genes, which can result in neuronal energy metabolism compensation defects and mitochondrial oxidative stress damage27. So the association of HIF1A gene polymorphisms (rs10873142, rs11549465, rs11549467) and the IS risk was explored in this study, which can put forward some strategies for the prevention and treatment of IS from genes perspective, it can also provide some reference for further research in the relationship between HIF1A gene polymorphism and IS.

Materials and methods

HIF1A SNPs screening

HIF1A SNPs were obtained from the National Center for Biotechnology Information (NCBI) database of SNP database (https://www.ncbi.nlm.nih.gov/snp/). HIF1A SNPs screening criteria were as follows: the human was selected as gene species; the gene frequency was greater than 5%; SNPs was validated by published literature; SNPs have been published by the 1000 Genomes Project.

Study population and sample collection

The association between HIF1A gene polymorphisms and IS was explored, the research workflow is shown in Fig. 1. Firstly, the study population were enroll from the physical examination center (n = 141, 54.6% male) and the Department of Neurology (n = 159, 63.5% male) in the Fourth People’s Hospital of Yunnan Province from March 1, 2022 to October 31, 2022. The inclusion criteria of IS group were all patients who lived in Yunnan for a long time, diagnosed with IS for the first time and 18 years old or older. Patients with connective-tissue disease, vasculitis, acute myocardial infarction, atrial fibrillation, heart valve disease, severe kidney and liver injury, severe infection, coagulopathy and death during treatment were excluded in the IS group. The physical examination group inclusion criteria were regular physical examination personnel whose age and sex ratio were matched with the IS group, head CT had no obvious abnormalities and lived in Yunnan for a long time. People with IS, hemorrhagic stroke, neurological deficit disease, genetic disease, severe cardiovascular and cerebrovascular diseases were excluded in the physical examination group. Written informed consent was obtained from all participants. This study has been approved by the Medical Ethics Committee of the Fourth People’s Hospital of Yunnan Province (Ethics approval number: 2020) YX No. (170).

Fig. 1
figure 1

Flow chart of this study.

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The collection of participants information

The clinical information of participants was collected by the electronic medical system and the physical examination center reporting system of the Fourth People’s Hospital of Yunnan Province. The age, gender, smoking history, drinking history, body mass index, fasting blood glucose, blood lipid levels, platelet count, previous disease history and other information of all participants were collected. In the IS group, neurological examination scores were also collected during hospitalization.

Collection of blood samples

Peripheral elbow venous blood was collected from participants in fasting and resting state, 2.0 mL venous blood was mixed in the anticoagulant tube, then the relevant information and number of the participants was label in the anticoagulant tube. blood samples was centrifuged 20 min (1000 g/min), which can obtain plasma. Finally, all samples were stored at − 80 °C.

DNA extraction and genotyping

DNA was extracted from blood cells by DNA extraction kit, DNA extraction time and information are marked on each tube, then DNA concentration was measured by spectrophotometer. Based on the reference sequence of HIF1A SNPs in NCBI dbSNP database, Primer 5.0 software was used to design primers for HIF1A SNPs. HIF1A SNPs Primer was synthesis by Anhui General Biology Company, the purity of primers was verified by MALDI-TOF mass spectrometer. Genotyping was performed using ABI SNaPshot Multiplex Kit, the procedure is as follows: DNA was amplified by Multiplex PCR, then single-base extension reactions were performed. The extension products were denatured with Hi-Di formamide, then the products were separated and analyzed using ABI 3730xl genetic analyzer by capillary electrophoresis method. The data were automatically genotyped by Gene Mapper 6.0 software (peak throughput threshold was set at 500 RFU and standard deviation of SNP typing was less than 0.5 bp), all experimental procedures were performed according to the strict double-blind principle. To evaluate genotyping reliability, 5% DNA samples were randomly retested, the success rate of genotyping was 100% in this study. Primer series are shown in Tables 1 and 2.

Table 1 Primer sequences for PCR amplification.
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Table 2 Primer sequence of snapshot extension.
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Haplotyping analysis of HIF1A

There was a linkage effect among the SNPs, so the haplotype analysis method was used to analyze the association between HIF1A gene polymorphisms and IS risk. Firstly, the three SNPs of HIF1A were analyzed by online haplotype analysis platform (http://shesisplus.bio-x.cn/SHEsis.html), then the genotyping results of participants was obtained, haplotypes with frequencies below 3% were removed in the results, finally, a total of three haplotypes were obtained.

ELISA

The expression level of HIF1A was measured by ELISA. The detection required antibodies indicators were pre-coated in the kits, the kits is containing microplate. The experimental steps included adding sample, adding enzyme-linked antibody, washing plate, color reaction, terminating reaction, machine detection and calculating sample concentration.

GEO analysis of HIF1A

“Ischemic stroke” were imported into the GEO database (http://www.ncbi.nlm.nih.gov/geo), “Homo sapiens” was selected as the species, “Series” was selected as the item type, peripheral blood was selected as the sample type, array analysis expression was selected as the type, finally, 1 result was obtained, which was GSE16561. The relevant information of GSE16561 dataset was downloaded, then HIF1A gene expression was analysis.

eQTL analysis

eQTL analyze has a certain significance in the association analysis of gene polymorphisms and gene expression levels. Based on GTEx platform (https://www.gtexportal.org/), the association between HIF-1 A rs10873142 and HIF-1 A expression in tissues was analyzed. The study power of HIF1A rs11542465 and rs11542467 genotypes was low, so the relationship between their genotypes and HIF1A expression levels was not analyzed in this study.

Statistical analysis

SPSS 22.0 software was used to analysis all data. Set α = 0.05, when P < 0.05, it indicated that there was statistical difference between the two groups. The χ2 test was used to analyze measurement data, such as gender. Then normality test was performed, when the measurement data conformed to the normal distribution and the variance was homogeneous, the Student’s t-test was performed. The Student’s test t was used to analyze age, fasting blood glucose and lipid levels of study subjects. If the measurement data did not conform to the normal distribution, the Mann-Whitney U test was performed, the Mann-Whitney U test was used to analyze the association between HIF1A SNPs and clinical and laboratory parameters of IS patients. Bonferroni was used to correct the results of all statistical analyses involving the need for P value correction.

Results

Study population characteristics

The clinical characteristics of study population are summarized in Table 3. The mean age of IS patients (101 males and 58 females) was 62.654 ± 11.779 years old. The average age of physical examination group (77 males and 64 females) was 62.504 ± 9.7201 years old. Student’s t-test analysis showed that there was no significant difference in age between the IS group and the physical examination group. χ2 test analysis showed that there was no significant difference in gender between the IS group and the physical examination group. The height, weight, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), apolipoprotein B and blood platelet count of the two groups were analyzed by student’s t -test, the above data were not statistical differences between the two groups (P > 0.05). The fasting blood glucose of two groups was compared by student’s t-test, the fasting blood glucose of IS group was higher than the physical examination group, which was statistically differences (P < 0.05). The total cholesterol (TC), HDL-C and apolipoprotein A1 levels of two groups were compared by student’s t-test, which has shown that the above data were higher in the physical examination group, the difference has statistically significant (P < 0.05).

Table 3 Analysis of clinical characteristics of participants.
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HIF1A gene polymorphism

The three SNP loci of HIF1A were detected by SNaPshot genotyping technology in Yunnan population, the results showed that the three SNP loci of HIF1A had gene polymorphism. The results showed that HIF1A rs10873142 had three genotypes (CC, CT and TT), the genotyping peak map was shown in Fig. 2. The distribution frequency characteristics of the above three genotypes and alleles were shown in Fig. 3. The HIF1A rs11549465 also has three genotypes (CC, CT and TT), its genotyping peak map is shown in Fig. 4. The above three genotypes and allele distribution frequency characteristics are shown in Fig. 5. HIF1A rs11549467 has two genotypes (AG and GG), the genotype peak map is shown in Fig. 6. the distribution frequency characteristics of the above two genotypes and alleles are shown in Fig. 7.

Fig. 2
figure 2

Genotyping of HIF1A rs10873142, (A) represents TT genotype; (B) represents the CT genotype; (C) represents the CC genotype.

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Fig. 3
figure 3

The genotype and allele frequency distribution histogram of HIF1A rs10873142.

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Fig. 4
figure 4

Genotyping of HIF1A rs11549465, (A) representing the TT genotype; (B) represents CC genotype; (C) represents the CT genotype.

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Fig. 5
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The genotype and allele frequency distribution histogram of HIF1A rs11549465.

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Fig. 6
figure 6

Genotype of HIF1A rs11549467, (A) represents GG genotype; (B) represents the AG genotype.

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Fig. 7
figure 7

The genotype and allele frequency distribution histogram of HIF1A rs11549467.

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Association analysis of HF1A SNPs with clinical and laboratory parameters in IS patients

The association HIF1A SNPs with clinical and laboratory parameters of the participants was analyzed, the results are shown in Table 4. which shows that the HIF1A SNPs genotyping is correlated with the clinical and laboratory parameters of the IS patients. The IS patients TC expression level was related to the HIF1A rs10873142 CT and TT genotypes, HIF1A rs11549456 CC genotypes and HIF1A rs11549467 AG and GG genotypes (P < 0.05). HDL-C expression level was associated with HIF1A rs10873142 three genotypes, rs11549456 CC and CT genotypes and rs11549467 GG genotypes (P < 0.05). The expression level of Apolipoprotein A1 was associated with all genotypes of the HIF1A three SNPs (P < 0.05). The expression level of ROS was associated with the CT and TT genotypes of rs10873142, the CC genotype of rs11549456 and the GG genotype of rs11549467 (P < 0.05). In this study, the association the HIF1A rs11549465 TT genotype with the clinical and laboratory parameters of IS patients was not analyzed, because the HIF1A rs11549465 TT genotype was less (Physical examination group: 1 case; IS group: 1 case).

Table 4 Association analysis of HF1A SNPs with clinical and laboratory parameters in IS patients.
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Association of HIF1A gene polymorphism and IS risk

Binary logistic regression analysis was used to analyze the association between HIF1A gene polymorphisms and IS risk. Firstly, polygenic models were constructed, which include co-dominant model, dominant model, recessive model, super-dominant model and allele model. The results are shown in Table 5. Bonferroni correction was used to correct the results of the above statistical analysis. When P´ < 0.0083, the results of HIF1A rs10873142 and rs11549456 has statistically significant. When P´ < 0.025, the results for HIF1A rs11549467 has statistically significant. Bonferroni correction analysis showed that the association between all genotypes and the IS risk was not statistically significant in the all genetic models of HIF1A rs10873142, rs11549465 and rs11549467. Then the power was calculated by the Hsieh-Fleckens formula, the result showed that the CT + TT genotype (68.2%) of the HIF1A rs10873142 dominant model and C allele (63.5%) of HIF1A rs10873142 allele model had moderate power, the power of remaining genotypes was less than 40% in the HIF1A rs10873142 genetic models. The power of all genotypes was less than 20% in the HIF1A rs11549465 and rs11549467 genetic models. The sample size of this study was relatively small, so the power of this study was small, in the future, research power can be improved through larger sample size studies.

Table 5 Analysis of HIF1A gene polymorphisms and IS susceptibility.
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Table 6 Analysis of the relationship between HIF1A haplotypes and the risk of IS.
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Haplotype analysis of HIF1A gene polymorphisms

The three haplotypes of HIF1A were obtained by haplotype analysis method, which were TCG, CCG and CTG, the results are shown in Table 6. The frequency of TCG haplotype was the highest, the frequency of physical examination group was 79.0%, the frequency of IS group was 71.5%, and it may reduce IS risk (36.0%) (P = 0.024), which is a protective factor for IS. The frequency of CCG haplotype was lower than TCG haplotype, the frequency of physical examination group was 13.1%, the frequency of IS group was 22.8%, and it may increase IS risk (99.6%) (P = 0.002), which is a risk factor for IS. the study power of HIF1A haplotypes was calculated by the Hsieh-Fleckens formula,, the result showed that the power of HIF1A CCG haplotype was 86%, the power of HIF1A TCG haplotype was 62%, the power of HIF1A and CTG haplotype was less than 20%, therefore, the association of HIF1A CCG haplotype and HIF1A TCG haplotype with IS could be further study in the future.

Analysis of HIF1A expression levels

HIF1A expression levels in peripheral plasma was measured by ELISA. The analysis results showed that the HIF1A expression level of participants did not conform to the normal distribution, so the Mann-Whitney U rank sum test was performed, the results are shown in Fig. 8A. which show that IS patients HIF1A expression level was 4.0184 (3.3916, 5.0089) pg/mL in the peripheral blood plasma, the HIF1A expression level of physical examination group was 0.45776 (0.33626, 0.76733) pg/ml in the peripheral blood plasma, there was significant difference between the two groups (P < 0.05).

Fig. 8
figure 8

HIF1A gene expression analysis: (A): ELISA results; (B): GEO results.

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GEO analysis results showed that HIF1A expression level is conform to the normal distribution in GSE16561 data set, so Student’s t test was performed, the results are shown in Fig. 8B. The results show that the HIF1A expression level of IS patients was higher in the peripheral blood plasma, which was 4.0184 (3.3916, 5.0089) pg/mL, the HIF1A expression level of physical examination group was lower in the peripheral blood plasma, which was 0.45776 (0.33626, 0.76733) pg/mL, there was significant difference between the two groups (P < 0.05), it shows that the HIF1A expression level of IS patients is higher than physical examination group in the peripheral blood plasma.

The relationship analysis between HIF1A rs10873142 and HIF1A expression level

The eQTL analysis results showed that HIF-1 A rs10873142 may affect the HIF-1 A expression level in tibia, lymphocytes, skin, esophagus and other tissues, however, HIF-1 A rs10873142 was not associated with HIF-1 A expression level in blood and brain tissues. The eQTL analysis results are shown in Fig. 9.

Fig. 9
figure 9

HIF1A rs10873142 eQTL analysis.

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Discussion

Participants clinical information were analyzed, the results showed that diabetes can increase IS risk. It has been reported that long-term hyperglycemia may stimulate oxidative stress response, which will hinder the vascular endothelial cells repair and stimulate blood to hypercoagulable state, which will lead to thrombosis, then it will lead to the narrowing or blockage of cerebral vessels28,29, at the same time, the body will undergo oxidative stress response and produce a large number of oxygen free radicals, which will lead to the expression of HIF1A increased. The increased HIF1A will regulate the level and function of various hormones, which will lead to the disorder of biological function of the body. Therefore, HIF1A may be associated with the pathological process of IS.

In recent years, studies have found that HIF1A gene polymorphism is associated with a variety of diseases, for example, HIF1A gene polymorphism may increase chronic obstructive pulmonary disease risk in the Chinese Han population24 and the incidence of stable strain angina pectoris30. In this study, the association between HIF1A genetic polymorphisms and IS was explored, the results showed that the HIF1A three SNPs genetic polymorphisms were not associated with the IS risk, but their genotype expression could affect the expression levels of clinical and laboratory parameters in IS patients, therefore, the HIF1A three SNPs genetic polymorphisms may be associated with the IS pathological process.

HIF1A gene polymorphism are related to genetic factors, which is involved in the pathological process of various diseases, such as chronic obstructive pulmonary disease, brain injury, diabetic retinopathy and various cancers31,32,33. IS has complex pathogenesis, multiple factors and multiple gene loci is related the pathogenesis of IS, so the further study of theses factors and these loci can better prevent and treat IS. After IS occurrence, a large number of HIF1A are activated under the state of hypoxia and ischemia. Then the SNPs of HIF1A will become very active, the each haplotype of these SNPs is play different roles34. HIF1A haplotype was analyzed by SNaPShot method in this study, SNaPShot method has the advantages of accurate typing, high detection success rate, high throughput and fast detection speed, and it is not limited by SNP polymorphisms and samples number. Study results showed that HIF1A TCG haplotype may be a protective factor for IS, which can reduce IS incidence. HIF1A CCG haplotype may increases IS incidence. So the risk of IS can be effectively predicted by further study of HIF1A SNPs haplotype.

It has been found that people with IS family history has greatly IS risk, which is due to genetic inheritance. At the same time, some gene mutations can also increase IS risk35,36. The results of this study showed that the HIF1A expression level of IS patients was higher than normal people. HIF1A is a master hypoxia response regulator, which is very sensitive to oxygen concentration, it can participate in energy metabolism, angiogenesis, apoptosis and cell proliferation, which plays an important role in the pathological process of IS. When the brain is hypoxic, HIF1A is activated in order to adapt to the hypoxic environment, which will activate many biological factors to regulate some biological functions, which can protect brain tissue and neurons, so it can participate in the pathophysiological process of IS. Therefore, the degree of cerebral hypoxia and damage can be estimated by monitoring the expression level of HIF1A.

The relationship between HIF1A rs10873142 and HIF1A expression level was explored, the results showed that HIF1A rs10873142 may affect the HIF1A expression level in some tissues. According to relevant literature, HIF1A is very sensitive to oxygen concentration, it can maintain body oxygen balance by enhancing HIF1A activity. When the brain is hypoxic, a large number of HIF1A are activated and their expression is increased, which can induce the expression of Bcl-2 interacting protein 3 (BNIP3), Over-expression BNIP3 may increase Becline-1 expression, which will trigger cell autophagy and inhibition mTOR pathway, then it will play a role in protecting nerve cells, which can improve the survival rate of neurons, reduce the size of cerebral infarction and reduce IS damage. Therefore, the pathological process of IS can be evaluated by monitoring the HIF1A expression and HIF1A genetic polymorphism, which can help doctor to design a individualized treatment plan.

Conclusion

The HIF1A gene polymorphism and the HIF1A expression level are closely related to the IS. The this study found that the genotype of HIF1A SNPs can affect the expression levels of clinical and laboratory parameters in IS patients, such as HDL-C, apolipoprotein A1, ROS. HIF1A TCG haplotype is a protective factor for IS, HIF1A CCG haplotype is a risk factor for IS. However, the association between HIF1A rs10873142, rs11549465, rs11549467 genotypes and the IS risk was not statistically significant in this study. HIF1A expression of IS patients is higher than the healthy controls. The study power of HIF1A CCG haplotype was 86%, the association HIF1A CCG haplotype with IS could be further study in the future. Therefore, IS pathological processes can be predicted by monitoring the HIF1A gene polymorphism and the HIF1A expression level, IS risk can be predicted by monitoring the HIF1A gene haplotypes.

Limitation

Potential limitations of this study warrant careful consideration. Firstly, it was a hospital-based case-control study, therefore, selection bias is inevitable and the subjects are not fully representative for the general population. Secondly, only HIF1A three SNPs were studied in this study, it is necessary to study the effect of the HIF1A other SNPs to the disease in the future. Thirdly, the sample size was relatively small in this study, so the statistical power of this study was greater than 80% only for the HIF1A CCG haplotype, therefore, large sample sizes studies need to be performed in the future.