Underexpression of SRSF3 and its target gene RBMX predicts good prognosis in patients with head and neck cancer

Abstract

Head and neck cancer collectively is one of the most common cancer types in the world. Oral squamous cell carcinoma (OSCC) is the most common subtype of head and neck cancer. SRSF3 is a protooncogene and is overexpressed in patients with OSCC. However, the relationship between SRSF3 expression and the clinical outcomes of patients with head and neck cancer remains unclear. By using the cBio-Portal for Cancer Genomics, a public online tool originally developed at Memorial Sloan Kettering Cancer Center (New York, NY, USA), it was revealed that patients with head and neck cancer with an underexpression of SRSF3 showed better overall and disease-/progression-free survival rates. Moreover, 227 genes were found to be significantly coexpressed with SRSF3 in head and neck cancer. Then, in combination with the analysis of a previous splice-array dataset that included significantly changed genes after the silencing of SRSF3, four potential target genes of SRSF3 were identified. RBMX and HNRNPL were further confirmed to be target genes of SRSF3. Moreover, the underexpression of RBMX was determined to be significantly associated with a favorable overall survival rate among patients, while patients with an underexpression of both SRSF3 and RBMX is a subgroup of individuals with better prognoses than all other patients. These results suggest that the underexpression of SRSF3 and that of its target RBMX can be used as potential biomarkers to predict favorable overall survival among head and neck cancer patients. Keywords; head and neck cancer, RBMX, SRSF3 Materials and Methods Cell culture and RNA interference CAL 27 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco Laboratories, Gaithersburg, MD, USA) and 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA). Separately, SCC-9 cells were cultured in a mixed medium of DMEM and Ham's F12 medium (1:1) supplemented with 10% FBS and 1% antibiotic-antimycotic solution and 400 ng/mL of hydrocortisone. Human SRSF3 small interfering RNA (siRNA) was synthesized by GenePharma (Shanghai, China). Cells were transfected with 20 nM of siRNA using lipofectamine 3,000 reagent (Invitrogen) according to the manufacturer's instructions. Forty-eight h later, cells underwent a second transfection and, 96 h after that, total protein and RNA samples were collected. The sequences of siRNAs used in this study are as follows: 5' AGAGCUAGAUGGAAGAACA 3' (SRSF3) and 5' ACUCUAUCUG-CACGCUGAC 3' (nonspecific; NS). Plasmid and transfection The T7-tagged SRSF3 expression plasmid (T7-SRSF3) was kindly provided by Dr. Zheng Zhi-Ming (National Cancer Institute, Bethesda, MD, USA). CAL 27 or SCC-9 cells were transfected with plasmid using lipofectamine 3,000 reagent according to the manufacturer's instructions. Western blot Total protein samples were loaded onto 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. The membrane was blocked by 5% nonfat milk and analyzed by the followi...

The Identification of Critical m6A RNA Methylation Regulators as Malignant Prognosis Factors in Prostate Adenocarcinoma

“…HNRNPG, also known as RBMX, is an m 6 A reader that preferentially binds non-coding RNAs and also binds m 6 A on mRNAs for their splicing (Zhou et al, 2019). Literatures exist showing that RBMX predicted prognosis in patients with head and neck cancer (Guo et al, 2020) and regulated apoptosis in breast cancer (Martinez-Arribas et al, 2006). The function of RBMX in PC remains unknown.…”

RNA packaging into extracellular vesicles: An orchestra of RNA‐binding proteins?

“…Functions Involvement in disease hnRNPA2B1 -Alternative splicing (Martinez-Contreras et al, 2007) -mRNA transport (Han et al, 2010) -Pri-miRNA maturation (Alarcón et al, 2015) -Telomere maintenance (Moran-Jones et al, 2019) -Innate immunity against DNA viruses (Wang et al, 2019) -Recognized as autoantigen in RA and SLE (Wu et al, 2018) -Prion-like properties in ALS, FTD, AD (Harrison & Shorter, 2017) -Overexpressed in lung cancer, pancreatic adenocarcinoma, breast cancer (Barceló et al, 2014;Hung et al, 2017;Klinge et al, 2019) hnRNPC1 -Formation of RNP complex with pre-mRNA (Rech et al, 1995) -Splicing regulation (König et al, 2010;Venables et al, 2008) -Transport of mRNA and snRNAs (Mccloskey et al, 2012) -Translation enhancer (Cieniková et al, 2015) -hnRNPC1 targets pri-miR-21 in glioblastoma multiforme, controlling its metastatic potential (Park et al, 2012) -Negative prognostic factor in advanced gastric cancer (Huang et al, 2016) hnRNPG (RBMX) -Alternative splicing (Heinrich et al, 2009) -Chromosome integrity and repair (Matsunaga et al, 2012) -Chromosome segregation (Cho et al, 2018) -Genome stability (Munschauer et al, 2018) -RBMX somatic mutations and altered expression levels associate with lung, endometrial and other solid tumours (Elliott et al, 2019) -Overexpression is associated to poor prognosis in oral squamous cell carcinoma (Guo et al, 2020) hnRNPH1 -Alternative splicing (Alkan et al, 2006) -DNA replication (Barrack, 1987) -RNA transcription (Barrack, 1987) -Inhibition of cell differentiation (Liu et al, 2001) -Overexpression associates with poorly differentiated solid tumours (head, neck, hepatocellular, pancreatic, colon, laryngeal, oesophageal, and prostate) (Sun et al, 2016) -hnRNPH1 mRNA in exosomes was suggested as a biomarker for HCC (Xu et al, 2018) and advanced prostate cance...…”

IQGAP1 and RNA Splicing in the Context of Head and Neck via Phosphoproteomics

“…Another investigation of HNSCC cell lines and tissues found MATR3, an RNA-binding protein and regulator of alternative splicing, to be upregulated in HNSCC. , Additionally, SRSF5, a splicing factor involved in constitutive splicing that can modulate the selection of alternative splice sites, has been shown to be upregulated in HNSCC tumors . Lower expression of SRSF9, another splicing factor similar to SRSF5, and RBMX, a protein implicated in alternative splicing that interacts with SRSF10, correlates with improved survival in HNSCC patients. − Searching the CPTAC HNSCC data also revealed that many of these splicing-related proteins have significantly altered phosphorylation levels between HNSCC tumors and normal tissue . Two of the significantly changing phosphosites in our NOKs data set (S853 in PRPF40B and S1424 in SRRM2) were significantly changed in the same direction between tumor and normal tissue as in the NOKs and NOKs IQGAP1KO (without significant change in the proteins).…”