title: [RNPC1 induces sensitivity of HER-2-positive breast cancer BT474 cells to trastuzumab through upregulation of HER2].
abstract: OBJECTIVE
  To explore the sensitivity of HER-2-positive BT474 breast cancer cells to trastuzumab after knockdown or overexpression of RNA binding protein 38 (RNPC1).
  
  
  METHODS
  The expressions of RNPC1 and HER-2 mRNA were detected by qRT-PCR, and the expressions of RNPC1, HER-2 and PI3K/AKT proteins were detected by Western blot after transfected with RNPC1 lentiviral vector, respectively. The experimental groups were treated with different concentration of trastuzumab, and cell apoptosis rate was analyzed by 7-AAD/APC double staining flow cytometry, and cell growth inhibition rate was tested by cell counting kit 8 (CCK-8). The expression of apoptosis-related proteins was detected by Western blot assay.
  
  
  RESULTS
  The results of qRT-PCR showed that overexpression of RNPC1 increased the expressions of RNPC1 and HER-2 mRNA, and the expressions of RNPC1 and HER-2 were decreased after RNPC1 knockdown. The knockdown of RNPC1 decreased the expressions of RNPC1 and HER-2. Moreover, overexpression of RNPC1 decreased and knockdown of RNPC1 increased the levels of p-PI3K and p-AKT while the total protein expressions of both were marginally changed.The results of analysis using a cell counting CCK-8 kit showed that the RNPC1 overexpressed group had a higher growth inhibition rate [(20.33±1.25)%, (35.38±2.05)%, (50.43±2.12)%, (65.35±2.08)% and(76.00±2.16)%, respectively] than that of the control group [(13.67±1.24)%, (27.86±2.05)%, (39.72±1.69)%, (53.33±1.70)% and(62.68±2.07)% ] when treated with different concentrations of trastuzumab (5, 10, 15, 20 and 25 μg/ml). The cell apoptosis rates in the RNPC1-overexpressed group [(19.46±1.06)%, (30.87±0.98)%, (50.45±1.13)%, respectively] were also increased compared with that in the control group [(14.38±0.64)%, (21.65±1.24)%, (38.03±0.85)%] when treated with different concentrations of trastuzumab (0, 10, 20 and 30 μg/ml)(P<0.05 for all). Reverse results were observed in the RNPC1 knockdown experiments [experimental groups: (9.67±1.18)%, (21.67±1.23)%, (30.33±1.25)%, (40.33±1.69)%, and (53.00±1.63)%] compared with those of control groups: [(14.00±0.82)%, (27.67±1.25)%, (39.67±1.79)%, (53.67±1.50)%, and (63.33±1.52)%]; and experimental groups: [(11.64±0.68)%, (16.60±1.01)%, and (25.14±3.12)%] compared with those of the control groups: [(14.71±0.61)%, (22.65±0.96)%, and (39.03±0.85)%]. The overexpression of RNPC1 increased the expression levels of Bim and Bad and decreased the level of Bcl-xl, and reverse result was observed after knockdown of RNPC1.
  
  
  CONCLUSIONS
  RNPC1 may promote the sensitivity of breast cancer cells to trastuzumab through the increased expression of HER-2 in the BT474 breast cancer cells.
authors.affiliation: Department of Breast Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
authors.forename: C L
authors.initials: CL
authors.lastname: Li
authors.forename: X J
authors.initials: XJ
authors.lastname: Zhou
authors.forename: P P
authors.initials: PP
authors.lastname: Lou
authors.forename: T S
authors.initials: TS
authors.lastname: Xia
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: Q
authors.initials: Q
authors.lastname: Ding
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020893:Receptor, ErbB-3 / Q000378:metabolism*; D015398:Signal Transduction; D000068878:Trastuzumab / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C515435:RBM38 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D020893:Receptor, ErbB-3; D000068878:Trastuzumab