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Free PDF Download European Review for Medical and Pharmacological Sciences 2021; 25: 3226-3234 LncRNA PRNCR1 aggravates the malignancy of oral squamous cell carcinoma by regulating miR-326/FSCN1 axis D.-K. LIU1, Y.-J. LI2, B. TIAN3, H.-M. SUN4, Q.-Y. LI5, B.-F. REN6 1Department of Stomatology, Yantaishan Hospital, Yantai, Shandong Province, China 2Department of Emergency Internal Medicine, Qingdao Municipal Hospital, Qingdao, Shandong Province, China 3Department of Clinical Laboratory Medicine, People’s Hospital of Rizhao, Rizhao, Shandong Province, China 4Hospital Infection-Control Department, Zhangqiu District People’s Hospital, Jinan, Shandong Province, China 5Imaging Center, Zhangqiu District People’s Hospital, Jinan, Shandong Province, China 6Medical Insurance Department, Zhangqiu District People’s Hospital, Jinan, Shandong Province, China Dekuan Liu and Yongjun Li contributed equally Abstract. – OBJECTIVE: The important regu- Introduction latory mechanism of lncRNA PRNCR1 has been emphasized in malignant tumors. However, the In recent years, oral cancer has been threaten- role of lncRNA PRNCR1 remains unclear in oral ing our health and is one of the most serious oral squamous cell carcinoma (OSCC). The purpose diseases. Oral cancer is more common in men, of this study is to reveal the role of lncRNA PRN- and the most common is oral squamous cell car- CR1 in OSCC. 1 PATIENTS AND METHODS: RT-qPCR was cinoma (OSCC) . The etiology of oral cancer is used to detect mRNA expression. The function- unclear and may be related to long-term smoking al mechanism of lncRNA PRNCR1 in OSCC was and alcohol abuse, poor oral hygiene, malnu- investigated by CCK-8, transwell and Luciferase trition, leukoplakia and erythema2. Oral cancer reporter assays. should be easier to detect than cancers elsewhere, RESULTS: LncRNA PRNCR1 was upregulat- but this is not the case. Taking the most common ed in OSCC and promoted cell proliferation, mi- tongue cancer among oral cancer as an example, gration and invasion. LncRNA PRNCR1 directly 3 binds to miR-326. The mutual inhibition between stage I patients account for only 10.9% to 25.4% . the expressions of lncRNA PRNCR1 and miR- In addition, the 5-year survival rate of OSCC pa- 326 was identified in OSCC. In addition, miR-326 tients with early oral cancer can reach more than restrained cell proliferation, migration and inva- 60%4. Therefore, early detection and treatment sion in OSCC. Further, miR-326 directly targets are very important for OSCC patients. FSCN1. FSCN1 expression was positively regu- Long non-coding RNAs (lncRNAs) have been lated by lncRNA PRNCR1 in OSCC. And FSCN1 proven to be important regulators of various dis- promoted the progression of OSCC and aggra- eases, including cancers, and their key roles have vated the carcinogenic effect of lncRNA PRN- CR1 in OSCC. also been determined in OSCC. In particular, ln- CONCLUSIONS: LncRNA PRNCR1 promotes cRNA KCNQ1OT1 promoted cell migration and the progression of OSCC by functioning as a inhibited apoptosis by regulating the miR-185-5p/ miR-326 ‘sponge’ to upregulate FSCN1 expres- Rab14 axis in OSCC5. LINC01234 facilitated the sion. growth and invasiveness of OSCC by regulating the miR-637/NUPR1 axis6. LncRNA PRNCR1 Key Words: has been widely reported to participate in human PRNCR1, Oral squamous cell carcinoma, MiR-326, FSCN1. diseases. Notably, lncRNA PRNCR1 was upreg- ulated and played an oncogenic role in breast can- 3226 Corresponding Author: Baofeng Ren, MD; e-mail: [email protected] LncRNA PRNCR1 aggravates the malignancy of OSCC cer7. Upregulation of lncRNA PRNCR1 in col- willing to join the study and signed informed orectal cancer promoted cell proliferation and cell consent. Patients’ exclusion criteria: (1) patients cycle progression8. In addition, lncRNA PRNCR1 failed to cooperate with researchers; (2) patients has been reported to promote the development who were diagnosed with multiple diseases; (3) of eclampsia by regulating the MAPK signaling patients who were treated before admission. pathway9. However, the regulatory mechanism of There were 14 cases of T1, 11 cases of T2, 12 lncRNA PRNCR1 in OSCC is still unclear and cases of T3, and 22 cases of T4. According to needs to be investigated. clinical Tumor Node Metastasis (TNM) staging, LncRNAs participates in tumorigenesis by there were 5 cases in stage I, 18 cases in stage II, acting as a ‘sponge’ of miRNA. Here, miR-326 and 12 cases in stage III. In addition, according was found to have a binding site with lncRNA to the squamous cell carcinoma staging method, PRNCR1. Abnormal expression of miR-326 has 13 cases were classified as highly differentiated been detected in breast cancer10 and glioma11. In squamous cell carcinoma, 7 cases as moderately addition, miR-326 has been proposed to act as a differentiated squamous cell carcinoma, and 15 tumor suppressor in human prostate cancer by cases as poorly differentiated squamous cell car- targeting Mucin112. Besides, lncRNA PCAT1 is cinoma. Tumor samples were obtained mostly a new serum-based biomarker that can enhance from the cheek areas of the mouth, and adjacent cell growth by sponging miR-326 in esophageal normal tissues were collected from the same pa- squamous cell carcinoma13. However, the rela- tient. All participants provided written informed tionship between lncRNA PRNCR1 and miR-326 consents. These tissues were frozen in liquid has not been confirmed in previous studies. nitrogen and stored at −80 °C. This study was It has been reported that lncRNA PRNCR1 can approved by the Institutional Ethics Committee interact with HEY2 to abolish miR-448-mediated of Yantaishan Hospital and performed following growth inhibition in non-small cell lung cancer14. the World Medical Association Declaration of In this study, we investigated how lncRNA PRN- Helsinki (“World Medical Association Declara- CR1 regulates fascin actin-bundling protein 1 tion of Helsinki: ethical principles for medical (FSCN1) in OSCC. Upregulation and carcinogen- research involving human subjects”, 2013). esis of FSCN1 have been found in gastric cancer and lung cancer15,16. LncRNA PCAT-1 has been Cells Culture reported to promote the tumorigenesis of prostate The human normal oral epithelial cell line cancer by regulating FSCN1 through miR-145- NHOK and the OSCC cell lines SCC-4 and 5p17. In addition, down-regulation of miR-326 has Cal-27 were obtained from the American Type been found to be associated with poor progno- Culture Collection (ATCC; Manassas, VA, USA). sis in gastric cancer patients and promoted the These cells were cultured in Dulbecco’s Modified growth and metastasis of gastric cancer by tar- Eagle’s Medium (DMEM) containing 10% FBS 18 geting FSCN1 . Therefore, the regulatory mech- (5% CO2, 37°C). anism of lncRNA PRNCR1/miR-326/FSCN1 was explored in OSCC. Cell Transfection PRNCR1 vector, PRNCR1 and FSCN1 siRNA, miR-326 mimics were purchased from GenePhar- Patients and Methods ma (Shanghai, China). Cal-27 cells grown at 70- 80% confluence were transfected with the vectors Clinical Tissues or oligonucleotides by using Lipofectamine 2000 Thirty-five patients diagnosed with OSCC (Thermo Fisher Scientific, Waltham, MA, USA). (mean age: 53 years, ranging from 28 to 77 years; 21 males and 14 females) without any RNA Isolation, Reverse Transcription and pre-operative radiotherapy or chemotherapy RT-qPCR were recruited. All histological diagnoses anal- Total RNA was extracted using TRIzol reagent yses were conducted in Yantaishan Hospital. (Invitrogen, Carlsbad, CA, USA). The PrimeS- All specimens were confirmed by pathological cript RT reagent kit (TaKaRa, Dalian, China) was examinations. Patients’ inclusion criteria: (1) used for first strand DNA synthesis. PrimeScript patients who were diagnosed as OSCC through RT reagent kit (TaKaRa, Dalian, China) and pathological biopsies; (2) patients who had a primers were used for RT-qPCR assay. GAP- complete medical record; (3) patients who were DH or U6 was used as an internal control. The 3227 D.-K. Liu, Y.-J. Li, B. Tian, H.-M. Sun, Q.-Y. Li, B.-F. Ren expressions of lncRNA PRNCR1, miR-326 and Results FSCN1 were quantified with the −∆∆cq2 method. The primers used in our work were as follows: LncRNA PRNCR1 Promotes Cell PRNCR1 forward, 5’-CCA GAT TCC AAG GGC Proliferation and Motility in OSCC TGA TA-3’, and reverse, 5’-GAT GTT TGG AGG Abnormal expression of lncRNA PRNCR1 CAT CTG GT-3’; miR-326, forward, 5’-GGC was observed in OSCC tissues and cells. It was GCC CAG AUA AUG CG-3’, reverse, 5’-CGT found that the expression of lncRNA PRNCR1 GCA GGG TCC GAG GTC-3’; U6, forward in OSCC tissues was higher than that in normal primer: 5’-CTC GCT TCG GCA GCA CA-3’, re- tissues (Figure 1A). In addition, upregulation of verse primer: 5’-AAC GCT TCA CGA ATT TGC lncRNA PRNCR1 was also detected in SCC- GT-3’; FSCN1 forward, 5’-CAC AGG CAA ATA 4 and Cal-27 OSCC cell lines compared with CTG GAC GGT-3’, reverse, 5’-CCA CCT TGT NHOK cells (Figure 1B). Due to the significant TAT AGT CGC AGA AC-3’; GAPDH forward, difference in the expression of lncRNA PRN- 5’-ACA TCG CTC AGA CAC CAT G-3’, reverse, CR1 in Cal-27 cells, Cal-27 cells were used for 5’-TGT AGT TGA GGT CAA TGA AGG G-3’. further experiments. In order to explore the role of lncRNA PRNCR1 in OSCC, PRNCR1 siR- Dual-Luciferase Reporter Assay NA was transfected into Cal-27 cells. We found The pmiR-GLO vector (Promega, Beijing, that the expression of PRNCR1 expression was China) with the 3’-UTR of wild-type or mutant significantly reduced by its siRNA (Figure 1C). PRNCR1 or FSCN1 was transfected into Cal-27 Additionally, knockdown of PRNCR1 inhibit- cells with miR-326 mimics. After 48 h, the Lu- ed cell proliferation (Figure 1D). Similarly, the ciferase activity was measured by the Dual-Lu- downregulation of PRNCR1 also inhibited cell ciferase reporter gene assay system (Promega, migration and invasion in Cal-27 cells (Figure 1E, Madison, WI, USA).
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