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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (1): 19-25. doi: 10.19723/j.issn.1671-167X.2025.01.004

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LIM and calponin homology domains 1 may function as promising biological markers to aid in the prognostic prediction of oral squamous cell carcinoma

Li XU1, Wen SHI2, Yuehua LI1, Yajun SHEN1, Shang XIE1, Xiaofeng SHAN1, Zhigang CAI1,*()   

  1. 1. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
    2. First Clinical Division, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
  • Received:2024-09-24 Online:2025-02-18 Published:2025-01-25
  • Contact: Zhigang CAI E-mail:c2013xs@163.com
  • Supported by:
    the National Natural Science Foundation of China(81902766)

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Abstract:

Objective: To explore the function of LIM and calponin homology domains 1 (LIMCH1) in the development and progression of oral squamous cell carcinoma (OSCC), along with their potential clinical applications. Methods: By utilizing transcriptome sequencing data from two groups of oral squamous cell carcinoma patients, along with bioinformatics analytical techniques such as Gene Ontology (GO) and gene co-expression networks, we identified genes that might play a pivotal role in the pathogenesis of oral squamous cell carcinoma. We employed real-time quantitative PCR and Western blotting to validate the expression patterns of these genes across twelve patient tissue samples. Furthermore, we conducted CCK-8 assays, flow cytometry analyses, and scratch wound healing assays to assess the impact of key genes on the biological behaviors of both the Cal27 oral squamous cell carcinoma cell line and the potentially malignant DOK oral lesion cell line. Additionally, we examined correlations between these key genes and clinical disease parameters in 214 oral squamous cell carcinoma patients using The Cancer Genome Atlas (TCGA) data; gene set enrichment analysis (GSEA) analysis results were also incorporated to enhance our findings from real-time quantitative PCR and Western blotting regarding potential mechanisms underlying the action of these key genes. Results: The integrated analysis of sequencing data and bioinformatics revealed that LIMCH1 exhibited significantly reduced mRNA (P < 0.001) and protein levels (P < 0.01) in the oral squamous cell carcinoma tissues compared with normal control tissues. In the Cal27 cells, the low LIMCH1 level group demonstrated a larger wound healing area within 24 hours than the control group (P < 0.01), enhanced proliferation capacity over 72 hours relative to the control group (P < 0.01), and an increased apoptosis rate within 24 hours compared with the high expression group (P < 0.05). However, no significant differences were observed between the low and high level groups in DOK cells. Furthermore, it was determined that low LIMCH1 level correlated with poor prognosis in the patients (P=0.013) and a higher lymph node metastasis rate (P < 0.05). Investigations into the potential mechanisms of action indicated that LIMCH1 did not influence the onset or progression of oral squamous cell carcinoma via the epithelial-mesenchymal transition pathway. Conclusion: LIMCH1 level may function as a promising biomarker to aid in the prognostic assessment of oral squamous cell carcinoma; however, its precise mechanistic role requires further investigation.

Key words: Oral squamous cell carcinoma, Oral potentially malignant disorders, LIM and calponin homology domains 1

CLC Number: 

  • R739.8

Table 1

The primers used in the research"

GenePrimers
LIMCH1Forward 5'-CAGACGCCTTCACCAGATGTA-3'
Reverse 5'-GATGAGGCAAGTCGGATTCAG-3'
GAPDHForward 5'-GGAGCGAGATCCCTCCAAAAT-3'
Reverse 5'-GGCTGTTGTCATACTTCTCATGG-3'
CDH1Forward 5'-CAGGCCTCCGTTTCTGGAAT-3'
Reverse 5'-GGTGTATACAGCCTCCCACG-3'
CDH2Forward 5'-AGGCTTCTGGTGAAATCGCA-3'
Reverse 5'-GGAGGGATGACCCAGTCTCT-3'
VimentinForward 5'-CTCTGGCACGTCTTGACCTT-3'
Reverse 5'-TTGCGCTCCTGAAAAACTGC-3'

Figure 1

Identification of key genes in the pathogenesis of oral squamous cell carcinoma A, tissue samples from two patients were collected for whole-transcriptome sequencing to screen for differentially expressed genes (DEGs) potentially linked to the malignant transformation of OSCC; B, the identified DEGs were used to construct a gene co-expression network based on the terms "squamous cell carcinoma" and "inflammation"; C, overlapping genes from both networks were considered preliminary candidates, Gene Ontology (GO) analysis of these candidate genes revealed their molecular functions, a heat map illustrates the expession levels of the ten most significantly enriched genes in the two samples; D, LIMCH1 emerged as the final candidate due to its strong correlation with the most significant function-actin cytoskeleton organisation."

Figure 2

Expression of LIMCH1 in TCGA data and expression trend in clinical samples A, the expression of LIMCH1 in OSCC was significantly lower than that in normal adjacent mucosa; B, consistent with this, analysis of TCGA data confirmed LIMCH1 downregulation in OSCC tissues ***P < 0.001); C, D, validation of this trend using clinical patient samples via RT-qPCR and Western blotting further demonstrated a significant decrease in LIMCH1 expression in OSCC tissues compared with that in normal adjacent mucosa in all 12 pairs of patient samples (**P < 0.01, ***P < 0.001).LIMCH1, LIM and calponin homology domains 1; RPKM, reads per kilobase per million mapped reads; Nor, normal; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase."

Figure 3

The relationship between LIMCH1 expression levels and the clinical disease characteristics of the patients A, the survival rate did not show a significant difference between patients with high and low LIMCH1 expression within the first two years post-surgery (P=0.109), however, beyond the the two-year mark, patients with low LIMCH1 expression exhibited a significantly lower survival rate compared to those with high LIMCH1 expression (P=0.013); B, a total of 214 patients were categorised into four groups based on their T stage, and LIMCH1 expression was compared across groups, revealing no significant correlation with tumor size (T1 n=19; T2 n=73; T3 n=46; T4 n=76); C, when classified by lymph node status, patients with metastasis (N1+N2+N3, n=116) exhibited significantly lower LIMCH1 expression compared to those without metastasis (N0, n=80, *P < 0.05); D, LIMCH1 expression showed no significant association with smoking status (1=never smoked, n=55; 2=currently smoking, n=60; 3=quit smoking, quit time > 15 years, n=37; 4=quit smoking, quit time≤15 years, n=59; the number of patients who have quit smoking and the duration of quitting is uncertain is very small and is not included in the statistics); E, LIMCH1 expression showed no significant association with alcohol consumption habits (Yes, having a drinking habit, n=66; No, no drinking habit, n=38). ns, not significant."

Figure 4

The impact of variations in LIMCH1 level on the biological characteristics of Cal27 and DOK cells A to C, reduced LIMCH1 level enhanced the apoptotic capacity of Cal27 cells, while apoptosis in DOK cells remained largely unaffected (*P < 0.05); D, F, LIMCH1 overexpression significantly impeded the migratory capacity of Cal27 cells (**P < 0.01); E, G, Nevertheless, downregulation of LIMCH1 level in DOK cells exhibited a minimal impact on their migratory capacity; H, I, Additionally, diminished LIMCH1 level increased the proli-ferative capacity of both Cal27 and DOK cells, albeit to varying degrees (**P < 0.01). ns, not significant."

Figure 5

An investigation into the potential role of LIMCH1 in mediating the epithelial-mesenchymal transition (EMT) pathway GSEA revealed that genes related to low LIMCH1 expression were significantly enriched in EMT pathway within the Hallmark subset. However, real-time quantitative PCR and western blot analysis of key EMT pathway genes under altered LIMCH1 expression showed no significant differences."

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