LINC01128 expedites cervical cancer progression by regulating miR-383-5p/SFN axis
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Cervical cancer (CC), causing significant morbidity and mortality worldwide, is one of the most common gynecological malignancies in women. SFN has been reported as a potential prognostic marker with apparent high expression in tumors. Nevertheless, the function mechanism of SFN is not clear yet in CC.
The relative expressions of RNAs were detected by real-time quantitative PCR (RT-qPCR). Colony formation assay, EdU stained assay and CCK-8 assay were to check cell proliferation ability in CC. Flow cytometry and apoptosis related proteins analysis were used to measure cells apoptosis capacity. Luciferase reporter assay and RNA pull down assay were to verify the molecular mechanism.
SFN was highly expressed in CC tissues and CC cell lines compared with normal tissues and normal cell line. After interfering SFN, cell proliferation, migration and invasion ability was inhibited as well as cell apoptosis ability was promoted. In subsequence, miR-383-5p exhibited conspicuous low expression in CC tissues. And miR-383-5p was found to bind to SFN and have anti-cancerous effects in CC. Moreover, LINC01128 displayed remarkable high expression in CC tissues. Besides, LINC01128 shortage could reduce the expression of SFN at mRNA and protein levels. And the affinity between LINC01128 and miR-383-5p was verified. In the end, it was proved that LINC01128 could enhance cell proliferation, migration and invasion as well as inhibit cell apoptosis by binding with miR-383-5p and upregulating SFN.
LINC01128 expedited cells cellular process in CC by binding with miR-383-5p to release SFN.
KeywordsLINC01128 miR-383-5p SFN Cervical cancer
Cell counting kit-8
Endogenous competitive RNA
long non-coding RNA
Real-time quantitative PCR
Cervical cancer (CC) is the second leading cause of cancer-related deaths in women worldwide due to its high incidence and mortality [1, 2]. Although the treatment strategies for CC have made great progress, such as surgical resection, radiotherapy and chemotherapy, the long-term prognosis of patients with cervical cancer is still not satisfactory due to frequent postoperative recurrence and resistance to radiotherapy and chemotherapy . In recent years, molecular targeted therapies have significantly advanced the prognosis of many cancers, such as melanoma, breast, lung and prostate cancer [4, 5]. Nevertheless, at present, targeted therapy for the molecular mechanism of CC has not been promoted and popularized in the worldwide. Hence, further understanding of the molecular mechanism of the occurrence and development of CC will contribute to the development of more effective CC treatment.
Stratifin (SFN) has been reported to enhance lung adenocarcinoma development at an early stage  and correlate with poor prognosis of ovarian cancer patients . Moreover, low expression of SFN has also been found to indicate poor survival of esophageal squamous cell carcinoma sufferers . However, the role of SFN in CC was rarely mentioned. It has been well-established that microRNAs (miRNAs) induce translation inhibition or promotion of their target messenger RNA (mRNA) through base pairs at partial or complete complementary sites [9, 10, 11, 12]. Nowadays, related biological research indicated the important role of non-coding RNA in many cancers [13, 14, 15]. Among them, long non-coding RNAs (lncRNA) play crucial roles in a host of biological processes. And lncRNAs are capable of regulating the expression of genes in various biological functions . Among these non-coding RNAs, miRNAs and lncRNAs belong to two major groups. miRNAs are short RNAs that have a length of 21–25 nucleotides, whereas lncRNAs are long-stranded RNAs that have a length of 200 nucleotides [17, 18, 19, 20]. It has been confirmed that one of the mechanisms of lncRNAs is the physical binding of miRNAs to reduce the inhibition of miRNAs on their true target mRNAs [21, 22, 23]. Therefore, these lncRNAs are referred to as endogenous competitive RNAs (ceRNA) [24, 25]. Hence, we would search for the lncRNA that could free SFN form the regulation of its upstream miRNA.
Taken together, the purpose of this study was to explore the expression and action of SFN, and elucidate the action mechanism of SFN in CC in detail.
Patients and tissue samples
Between 2013 and 2018, 33 pairs of cervical cancer tissues and adjacent normal tissues were collected from patients who underwent surgery at The Second Xiangya Hospital, Central South University. What’s more, none of the patients in this study received chemotherapy or radiotherapy before surgery. Tissue specimens were frozen in liquid nitrogen at − 80 °C at the time of collection.
Cervical cancer cells (HeLa, SiHa, CaSki and C33a) and normal cervical cell (Ect1-E6E7) were purchased from the Cell Culture Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were all cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific). Cells were cultured with 5% CO2 at 37 °C.
HeLa or SiHa cells were plated in 6-well plates, and then incubated for one day. After that, the specific short hairpin RNAs (shRNAs) of SFN (sh-SFN-1/2) or LINC01128 (sh-LINC01128–1/2) and their negative controls (sh-NCs), along with mimics and inhibitor of miR-383-5p or miR-107-mimics, NC mimics/inhibitor were purchased from Genepharma (Shanghai, China). The above plasmids were separately transfected into HeLa or SiHa cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) after 48 h.
RNA extraction and real-time quantitative PCR (RT-qPCR)
Tissues and cells were lysed via Trizol reagent (Invitrogen), and then total RNA was extracted and reverse transcribed into cDNA via a Reverse Transcription Kit (Invitrogen). Then, RT-qPCR was conducted through SYBR Green RT-PCR Kit (Invitrogen) on an Applied Biosystems 7300 (Thermo Fisher Scientific). Results were calculated via the 2−ΔΔCt method. GAPDH/U6 level was measured as the internal control.
Cell counting kit-8 (CCK-8) and Colony formation assays
The number of HeLa or SiHa cells was 1000, which was seeded in 96-well plates and incubated under the standard conditions. CCK-8 solution (Dojindo, Tokyo, Japan) was added to each well. Then, cells were incubated for another 4 h. The absorbance at 450 nm was evaluated through a microplate reader (Olympus, Tokyo, Japan). For colony formation assay, HeLa or SiHa cells in medium (Thermo Fisher Scientific) were seeded in 6-well plates and incubated for two weeks. Colonies (≥50 cells) were formed and then manually counted.
5-ethynyl-2′-deoxyuridine (EdU) incorporation assay
EdU labeling medium was added to cell culture with EdU labeling kit (RiboBio, Guangzhou, China). HeLa or SiHa cells after transfection were incubated for 2 h, and then fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed at room temperature by PBS (Sigma-Aldrich) dyed with anti-EdU (Acbam, Cambridge, USA) working solution (Invitrogen). Under the same conditions, cells were again washed with Triton X-100 (Solarbio, Shanghai, China) in PBS (Sigma-Aldrich). Cells were observed via a fluorescent microscopy (Leica, Wetzlar, Germany).
This experiment was performed as previously described .
Transfected HeLa or SiHa cells were isolated via RIPA lysis buffer (Invitrogen). Then, the BCA (Invitrogen) was employed to evaluate the concentration of protein. Proteins were separated by SDS-PAGE (Millipore, MA, USA), followed by transferred into PVDF membranes (Millipore). After being blocked with 5% nonfat milk, membranes were incubated overnight with primary antibodies at 4 °C: anti-Bax (ab32503, Abcam), anti-Bcl-2 (ab182858, Abcam), anti-SFN (ab14123, Abcam) anti-E-cadherin (ab194982), anti-N-cadherin (ab202030), anti-Vimentin (ab193555), anti-ZEB1 (ab228986), anti-Slug (ab51772), anti-Twist (ab187008), anti-Snail (ab229701) and anti-GAPDH (ab8245, Abcam), then cultivation at room temperature with secondary antibody for 1 h. GAPDH was employed as the internal parameter. Finally, the ECL Kit (Thermo Fisher Scientific) was selected to visualize protein bands.
Transfected HeLa or SiHa cells were cultivated into 6-well plates. After being washed twice with cold PBS (Sigma-Aldrich) and re-suspended, cells were fixed through ice-cold ethanol (Sigma-Aldrich). Finally, flow cytometer (BD Biosciences, San Diego, CA, USA) was employed to analyze cell apoptosis.
The re-suspended cells in serum-free medium were seeded into the upper chamber, and the basolateral chamber was filled with 10% PBS. Transwell chambers pre-coated with or without Matrigel (Millipore, Massachusetts, USA) were employed to carry out the invasion or migration assay. An inverted microscope (Carl Zeiss, Jena, Germany) was applied to observe the migrated or invaded cells.
Sphere formation assay
HeLa or SiHa cells were harvested, counted and incubated in 6-well ultra-low attachment plates (Corning Incorporated, Corning, NY, USA) in serum-free RPMI 1640 medium (Invitrogen, Shanghai, China) to form sphere. The RPMI 1640 medium is supplemented with 20 ng/ml human FGF (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/ml human EGF (Gibco), 1% N2 supplement (Gibco) and 1% B27 (Gibco). 2 weeks later, the number of spheres was counted by a light microscope (Nikon Corporation) and the number was recorded.
Isolation and purification of cytoplasmic and nuclear RNA were conducted via a Cytoplasmic and Nuclear RNA Purification Kit from Norgen (Ontario, Canada). The expression levels of LINC01128, GAPDH and U6 were separately evaluated via RT-qPCR analysis.
RNA pull-down assay
miR-383-5p biotin probe and miR-383-5p no-biotin probe were treated with M-280 Streptavidin magnetic beads (Invitrogen) to generate probe-coated beads. Subsequently, cells were collected and dissolved, submitted to sonication and cultivation with probe-coated beads overnight at 4 °C. Lastly, RT-qPCR was employed to measure purified RNA complex.
Luciferase reporter assay
Partial DNA sequences of SFN or LINC01128 containing wild-type (WT) or mutant (MUT) miR-383-5p binding sites were amplified by PCR and then cloned into a pmirGLO dual-luciferase plasmid (Promega, Madison, WI, USA) to produce SFN-WT, SFN-MUT, LINC01128-WT, and LINC01128-MUT reporter plasmids, which were separately co-transfected with miR-383-5p mimics or NC mimics into HeLa or SiHa cells. Finally, luciferase activities were evaluated through the dual luciferase reporter assay system (Promega).
Results were presented as mean ± SD of three independent experiments at least. Statistical analyses were imported into SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Student’s t-test or one-way ANOVA was employed for analysis of differences. P < 0.05 had statistically significant.
The high expression of SFN promotes the cellular process of CC cells
Correlation between SFN expression and clinicopathological features. (n = 33)
< 4 cm
> 4 cm
Lymph node metastasis
SFN functions as a target gene of miR-383-5p
LINC01128 functions as miR-383-5p molecular sponge
LINC01128 accelerates proliferation and restrains apoptosis of CC cells by sponging miR-383-5p and targeting SFN
CC is one of the most common gynecological malignancies with significant morbidity and mortality in women. It is often reported that there is an urgent need to screen biomarkers that can be used for clinical diagnosis and treatment of CC patients due to the reasons of delayed diagnosis and poor treatment effect. In present study, we detected the expressions of SFN in CC tumor tissues and cells were much stronger. In addition, interfering SFN dramatically inhibited cells proliferation, migration, invasion and EMT process as well as promoted cells apoptosis. Thus, we inferred that SFN acted as an oncogene in CC cells.
Then, miR-383-5p was identified as the upstream gene of SFN. In the meantime, reports have shown inhibitory effects of miR-383-5p in adenocarcinoma [27, 28]. Besides, miR-383-5p acted as a tumor suppressor to get involved in the ceRNA mechanism [29, 30]. miR-383-5p was quantified to be downregulated in CC tissues. Additionally, we found that SFN was enabled to bind with miR-383-5p and be suppressed by miR-383-5p overexpression. Functionally, miR-383-5p overexpression could induce an inhibition in cell proliferation of CC cells.
The dysregulation of lncRNA was identified to be associated with the development of disease . Besides that, lncRNA plays an extremely significant role in various biological processes such as proliferation and apoptosis. Since several lncRNAs were found to bind and isolate miRNAs and block the role of miRNAs, we have speculated the presence of lncRNAs that combine and interdict with miR-383-5p and play a carcinogenic role in CC. And LINC01128 was screened out from Starbase to bind to miR-383-5p. Nevertheless, literature concerning LINC01128 is very limited. In this work, it was spotted that LINC01128 exhibited conspicuous high expression in CC tissues and cells. Moreover, LINC01128 shortage could suppress SFN level. Next, we discovered the location of LINC01128 in cytoplasm through nuclear cytoplasm separation experiment. Besides that, LINC01128 was verified to bind to miR-383-5p. Finally, it was proved that LINC01128 enhances the malignancy of CC dependent on miR-383-5p and SFN.
The major limitation of our study lies in the absence of in vivo assays, through which we could observe the influences of SFN on CC cells growth and metastasis. We would carry out in vivo assays in further investigation. Anchored in the above evidences, it could be concluded that LINC01128 poses promotional influences on cell proliferation, migration, invasion and EMT process in CC via sponging miR-383-5p and upregulating SFN. In other words, the LINC01128/miR-383-5p/SFN axis was conducive to enhance the CC progression.
In summary, the availability of miR-383-5p was antagonized by LINC01128 for the SFN-induced cell proliferation, migration, invasion and EMT process in cervical cancer, shedding a new light into understanding CC.
Sincerely thanks all the participants for their support.
YH designed the research; YH, YM and JL performed the experiments and analyzed the data; YC, MZ and XF drafted the manuscript. YH and YM checked the manuscript. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Written informed consent was obtained from all patients. Furthermore, this study got approval by the Ethics Committee of the Second Xiangya Hospital, Central South University.
Consent for publication
The authors declare that they have no competing interests.
- 7.Hu Y, et al. Expression profile and prognostic value of SFN in human ovarian cancer. Biosci Rep. 2019;39(5). https://doi.org/10.1042/BSR20190100.
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