Effect And Mechanism Of MiR-29c Targeting SERPINH1 On Oral Squamous Cell Carcinoma

Objective: Cancer is a multi-step process,in which cell mutations accumulate and lead to abnormal proliferation,migration,invasion and apoptosis.Oral cancer is a major public health problem in the world,ranking sixth among human malignant tumors.The 5-year mortality rate of OSCC is close to 50%,of which 90% is squamous cell carcinoma(OSCC),accounting for 43.8% of new HNSCC(head and neck squamous cell carcinoma)cases and 37.8% of deaths.Oral cancer is characterized by occult onset,difficult diagnosis,and rapid development,often accompanied by metastasis and damage treatment.In NCCN guidelines,surgery is recommended for patients with early-stage tumors,and surgery or definite concurrent chemo radiotherapy is recommended for patients with advanced tumors.However,the recurrence rate of OSCC is 30%,and local recurrence is the most common.Therefore,it is necessary to find and develop new strategies for accurate diagnosis and effective treatment of OSCC.The number of stromal cells(such as fibroblasts,macrophages and pericytes)increased with the progression of tumor.Serpin peptidase inhibitor clade H member 1(SERPINH1)belongs to the serpin superfamily,which is necessary for proper folding and secretion of collagen.The abnormal expression of SERPINH1 is closely related to a variety of cancers.In our current work,TCGA analysis showed that SERPINH1 was abnormally expressed in OSCC,but its specific regulatory mechanism is still unclear.To explore this,we analyzed the role of SERPINH1 in the occurrence and development of OSCC,and studied its effects on the growth and migration of OSCC cells.Identify the upstream micro RNA of it through bioinformatics analysis.Furthermore,we explored the molecular mechanism of SERPINH1 regulating by its micro RNA by cell biology experiments.Methods:1.GEO2 R analysis revealed differentially expressed genes in OSCC.2.The expression and prognosis of SERPINH1 in OSCC were analyzed by UALCAN data set.3.Western blot and real time PCR were used to study the expression of SERPINH1 in OSCC cell lines.4.HE staining was used to observe the pathology of clinical samples.5.The expression of SERPINH1 in OSCC samples was observed by immunohistochemistry.6.Western blot and real time PCR were used to study the protein and gene expression of SERPINH1 in OSCC samples.7.The effect of sh-SERPINH1 on the proliferation of OSCC cells was studied by MTT assay.8.The effect of sh-SERPINH1 on the migration of OSCC cells was observed by scratch test and transwell test.9.DAPI staining was used to observe the effect of sh-SERPINH1 on OSCC cell apoptosis.10.The effect of sh-SERPINH1 on OSCC cell cycle was observed by flow cytometry with PI staining.11.Targetscan bioinformatics database was used to predict the target and study the potential regulatory factors upstream of SERPINH1.12.Linkedomic database was used to study the correlation of miR-29 c in the occurrence and development of OSCC and the regulation of SERPINH1.13.The expression of miR-29 c in OSCC cell lines were detected by real time PCR.14.Luciferase double reporter gene detection was used to study the targeted regulation relationship between miR-29 c and SERPINH1.15.The effects of miR-29 c overexpression and SERPINH1 overexpression on the proliferation of OSCC cells were studied by MTT assay.16.The effects of miR-29 c overexpression and SERPINH1 overexpression on the migration of OSCC cells were observed by scratch test and transwell test.17.DAPI staining was used to observe the effect of miR-29 c overexpression and SERPINH1 overexpression on OSCC cell apoptosis.18.The effect of miR-29 c overexpression and SERPINH1 overexpression on OSCC cell cycle was observed by PI single staining flow cytometry.Results:1.GEO2 R analysis showed that SERPINH1 was highly expressed in OSCC.2.UALCAN data set analysis showed that SERPINH1 was highly expressed in OSCC and was associated with poor prognosis.3.Western blot and real time PCR showed that SERPINH1 was highly expressed in OSCC cell line.4.Immunohistochemistry showed that SERPINH1 was highly expressed in OSCC clinical samples.5.Western blot and real time PCR showed that SERPINH1 was highly expressed in OSCC.6.MTT assay showed that the down-regulation of SERPINH1 inhibited the proliferation of OSCC cells.7.Scratch test and transwell test showed that the down-regulation of SERPINH1 inhibited the migration of OSCC cells.8.DAPI staining showed that the down-regulation of SERPINH1 expression increased the apoptosis of OSCC cells.9.PI staining flow cytometry showed that the down-regulation of SERPINH1 expression increased the number of OSCC cells arrested in S phase.10.Targetscan bioinformatics database was used for target prediction,and miR-29 c was identified as the potential regulator of SERPINH1 upstream.11.Linkedomic database miR-29 c inhibits the occurrence and development of OSCC and negatively regulates SERPINH1.12.Real time PCR showed that miR-29 c was low expression in OSCC cell line.13.Luciferase double reporter gene detection showed that SERPINH1 was the target gene of miR-29 c,which was negatively regulated by miR-29 c in OSCC.14.MTT assay showed that miR-29 c overexpression inhibited the proliferation of OSCC cells,and SERPINH1 overexpression could reverse this inhibition.15.Scratch test and transwell test showed that miR-29 c overexpression inhibited the migration of OSCC cells,and SERPINH1 overexpression could reverse this inhibition.16.DAPI staining showed that overexpression of miR-29 c promoted the apoptosis of OSCC cells,and overexpression of SERPINH1 could reverse this effect.17.PI single staining flow cytometry showed that overexpression of miR-29 c led to the growth arrest of OSCC cells in S phase and inhibited the proliferation of OSCC cells.Overexpression of SERPINH1 could reverse this inhibitory effect.Conclusions:1.SERPINH1 was up-regulated in OSCC.2.Silencing SERPINH1 inhibited the proliferation,metastasis and invasion of OSCC cells,and increased the apoptosis of OSCC cells.3.Mi R-29 c can regulate the expression of SERPINH1.4.Overexpression of miR-29 c can inhibit the proliferation,metastasis and invasion of OSCC cells through SERPINH1,and increase the apoptosis of OSCC cells.