CircCASC15-miR-100-mTOR may Influence the Cervical Cancer Radioresistance

doi:10.21203/rs.3.rs-1036214/v1

PDF

Research Article

CircCASC15-miR-100-mTOR may Influence the Cervical Cancer Radioresistance

https://doi.org/10.21203/rs.3.rs-1036214/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Cervical cancer has ranked the top one in gynecological malignancies for incidence. Radioresistance is now becoming a leading reason of recurrence.

Methods

Our microRNA array data indicated that the miRNA-100 level decreased significantly during radioresistance. In this study, we up-regulated miR-100 in Hela and Siha cells by using miR-100 mimics and observed apoptosis, proliferation, cell cycle and invasion.

Results

It turned out that with overexpression of miR-100, the cells had more apoptosis and less invasiveness as well as proliferation. It may also influence cell cycle via target gene mTOR, and it deed reduced EMT. To examine the role of miR-100 in radioresistance, there was no significant result showed by BSP.While the circCASC15 has been identified with sponge function according to RNA pull down and ISH.

Conclusion

The conclusions indicate miR-100 is a tumor suppressor gene and could be a therapeutic target in radio-resistant cervical cancers.

Oncology

Molecular Genetics

Molecular Biology

cervical cancer

radio-resistance

miR-100

BSP

circCASC15

Radiotherapy (RT) has been widely applied from stage I to stage IV cervical cancer as primary or preoperative or postoperative adjuvant treatment. However, recurrence after radiotherapy remains significant. Approximately 20% of patients diagnosed with pelvic recurrence, while the cure rate for early-stage is more than 80% [1]. The 5-year survival rates of recurrenceis between 10.1% and 22.3% [2–3]. Therefore, elimination of RT was important for patients diagnosed with cervical cancer.

MicroRNAs (miRNAs) belong to non-coding RNA family that function at the post-transcriptional level. Their deregulated expression can influence both the treatment response[4] and the development of drug resistance [5–6].

MiR-100 belongs to miR-99 family, which located in chromosome 11. It has been proved playing a role in proliferation, metastasis, as well as sensitivity of to radiotherapy and chemotherapy [7–10].We proved miR-100 downregulated in radioresistant cervical cancer.

Circular RNA (circRNA) is another kind of non-coding RNA because it is a closed covalent loop without poly A tail or 5'to 3'polarity [11]. At present, more than 30,000 circular RNAs have been identified [12]. A variety of circular RNAs are believed to have relationship with occurrence of many tumors [13].Several reports have showed that miRNA response elements exist in circular RNAs, which act as competitive endogenous RNAs (ceRNAs) to sponge miRNAs or transcriptional regulators to. [14].We found circCASC15 may be the sponge of miR-100.

Patient selection and human tissues

Group 1 (for miRNA array)

Three patients have been clarified as stage IIB-IVA according to International Federation of Gynecology and Obstetrics (FIGO),, who had received radiotherapy in our hospital between 2000 and 2008. According to the 2013 NCCN cervical cancer clinical practice guidelines, patients received standard radiotherapy, pelvic radiotherapy and branch radiotherapy, with a total dose of 70 Gy at point A [15]. Two pathologists evaluated histology independently. According to the hospital's internal review and ethics committee, all samples were collected with informed consent.

Group 2 for QPCR. 84 cervical tumors were obtained from our department, between January 2002 and June 2011. None of themreceived any treatment before surgery. All patients have been confirmed HPV infection. Two patients without HPV infection were chosen as normal controls.

Mirnas Real Time Polymerase Chain Reaction Array

After isolation total RNA from samples, Universal cDNA synthesis kit (Exiqon) was used. MiRNAs were detected by the miRCURY LNA™ Universal real time microRNA polymerase chain reaction system(Exiqon, kangchen, China).

Cell Culture

HeLa and SiHa were purchased from China Academic Sinica Cell Repository, Shanghai, China. They were in MEM medium(Gibco, Los Angeles, CA, USA) with 10% fetal bovine serum (Gibco,Australia)in an incubator under humidity at 37℃with 5% CO₂.

Transient Transfection

Mir-100-5p mimics purchased from Genepharma (Shanghai, China), were transient transfected into cells with Lipofectamine™ 2000 (Invitrogen). Mock-transfected condition was made by negative control mimics with fluorescence. All the cells were collected at 48h post-transfection, and the levels of mir-100 were elevated by RT-PCR assay as follow. The sequences of miR-100-5p mimics and negative control were shown below:

Mimics(forward 5’-AACCCGUAGAUCCGAACUUGUG-3’

Reverse 5’-CAAGUUCGGAUCUACGGGUUUU-3’);

Negative control(forward 5’-UUCUCCGAACGUGUCACGUTT -3’

Reverse 5’-ACGUGACACGUUCGGAGAATT -3’);

Mirna Expression Analysis And Rt-pcr Assay

The RNAs were collected from cells by Trizol extraction (Invitrogen), and cDNAs were acquired using an RNA reverse transcription amplification kit (Takara). RT-PCR assays were performed by SYBR Green Real-time PCR Universal Reagent (GenePharma Co.,Ltd.) and analyzed by BIO-RAD fluorescence quantitative PCR machine. Primers were as follows:

miR-100-5p (5’-AACCCGTAGATCCGAACTTGTG-3’);

u6(5’-ACGCAAATTCGTGAAGCGTT-3’);

GAPDH(forward5’-TGGAAATGACAGTGAAGCACCTC-3’

reverse5’-TCGTTCATGCACTCGCTGAAG-3’);

u6 was used as control for miRNA, GAPDH was used as control for mRNA. Both of them was calculated with the 2 − ΔΔCT method.

Proliferation Assay

After cultured (1×10⁴ cells/well) in separate 96-well plates for 24h, cells were transfected with miR-100-5p mimics. At day 1, day 2, day 3, day 4 after transfection, cells activation were evaluated by Cell Counting Kit-8 assay, the absorbance were read at 450 nm.

Cell Apoptosis Assay

After digestion with EDTA-free trypsin, incubation with Annexin-V and PI for 10 min at20°C, flow cytometry was performed (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA)

Tumor Xenografts

Siha cells (2×10⁶) were transfected with miR-100 or Negative Control for 2 days following animal experiments institutional ethical guidelines. Cells were digested and resuspended in 200µL PBS .PBS with cells or without cells were injected into female BALB/c athymic nude mouse(four-week-old). Six nude mice were used. Mice were anesthetized with Isoflurane (Forene, Abbott GmbH & Co.KG, Wiesbaden, Germany) with 1 L*min^-1 O2 flow for euthanasia.

Bioinformatics

miRanda,TargetScan were chosen for predicting targetgene.

Immunohistochemistry

After embedded by paraffin- and fixed with formalin, tissue was cut into 5µm section, deparaffinization, rehydration, quenche, and retrieve.Incubation at 4°C overnight with Anti-mTOR (phospho S2448) antibody (HRP) (ab196914) (Abcam).

Establishment Of Radioresistant (Rr) Cell Lines

Cells were exposed to two Gy 3 times, four Gy 3 times, six Gy 3 times, eight Gy 2 times and ten Gy 2 times at 300 cGy/min with a linear accelerator (SIMEN, Germany). [16–17].

5-aza-2-deoxycytidine Treatment

HeLa and SiHa cells(3×10⁵cells/well) were cultured in 6-well plates overnight, then 10µM DAC(5-aza-2-deoxycytidine) or 10µM DMSO (dimethyl sulfoxide) was separately added to cells per 24h. Cells were harvested after 5 days, and RNAs were extracted for detecting the level of mir-100 as mentioned above.

Bisulfite Sequencing Pcr(Bsp)

Following extraction ( QIAamp DNA Mini Kit),Genomic DNA was subjected to EpiTect® Plus DNA Bisulfite Kit. After purifirationt, modified DNA was chosen as nested PCR reactions template. The primers were: outer primers, 5’-ATTCGAATTTAGTGGAATTAGAATC-3′ (forward) and 5′-AACCTACAACAACAACAACAACG-3′(reverse); nested primers, 5′-TTAGTA ATTTTAGGTTAGAGGGTTATCG-3′ (forward) and 5′-ACTCCAAAAACCC ATAACTAACCG-3′ (reverse). The second-round PCR products were subjected to electrophoresis on agarose gels, excised, and inserted into the TOPO cloning vector (Invitrogen, K4600-01).Clones were used for DNA sequencing randomly.

Western blotting

Protein was extracted from cells with ice-cold radioimmunoprecipitation lysis solution, then centrifugated to remove cell debris. N-cadherin (ab18203) and E-cadherin (ab194982) from Abcam(Cambridge, MA, USA) were chosen as primary antibodies and HRP-conjugated secondary antibody was purchased from Sangon (Shanghai, China).GAPDH and β-actin were employed as loading controls.

CircRNA expression profiles in CC

We enriched the contained circRNAs and digested them with RNase A, then reversely transcribed them to RNA by fluorescent reagents and random primers. We used Human circRNA Arrays (8 × 15 K, Arraystar, Rockville, MD, USA) to determine the circRNA profile. Those circRNAs with fold change ≥ 2 were identified as differentially expression circRNAs. The data were analyzed by using R software limma and Arraystar program (Arraystar).

Biotin-coupled probe pull-down assay

Hela and Siha cells were transfected by biotinylated miR-100-5p mimics or its mutant (Genepharma, Suzhou, China). Then cells underwent harvest, lysis, sonicate, and incubation with magnetic beads at 4℃ overnight. The RNA mix bound to the magnetic beads was eluted and treated with Trizol for further qRT-PCR.

Florescent in situ hybridization

Fluorescent in situ hybridization probes for circCASC15, miR-100, and 18S RNA were designed and synthesized by Synbio Technologies (Suzhou, China). 18S RNA was used as a positive control. The signals of the probe were examined by the Florescent in Situ Hybridization Kit (RiboBio, Guangzhou, China).

Data Analysis

All data were based onthree independent replicates. Statistical analyses were analyzed by SPSS 13.0. Data were presented as the mean values ± standard error. P<0.05 was considered statistically significant.

MiR-100 is down-regulated in radioresistant human cervical cancer.

To investigate miRNAs differential expressions in human radioresistant cervical cancer and to seek their roles in radioresistance, we performed miRNA microarray profiling analysis. At last we confirmed a set of miRNAs that are down-regulated in invasive cervical cancer. The most deregulated miRNA is miR-100 at 5 folds(P=0.028) (Fig. 1).

MiR-100 is correlated with poor clinical outcomes in cervical cancer.

The expression of miR-100 in 84 samples of cervical cancer and normal cervical tissues from hysterectomy including uterine myoma or prolapse.In Fig. 2, the average expression of miR-100 in cancer samples was lower than normal samples (P< 0.01). We found that miR-100 expression was correlated with tumor size (P<0.001), lymph node (LN) status (P<0.001) and TNM stage (P<0.001).

MiR-100 inhibits invasion and reduces EMT.

Since the fact that miR-100 was decreased at 5 folds in invasive cervical cancer according to the array, miR-100 mimics was used to up-regulate its expressions in Hela and Siha(Fig. 3A). Cell invasiveness was tested by transwell assay in 24-well-plate which illustrated that miR-100 could inhibit invasion at 60% in Hela and 50% in Siha cells (Fig. 3B). Wondering whether the inhibition of invasiveness was mediated via EMT, Western Blot was performed. As expected, up-regulation of miR-100 reduced EMT in protein levels(Fig. 3C).

MiR-100 promotes cell apoptosis and inhibits proliferation in cervical cancer.

The observed down-regulation of miR-100 in human invasive cervical cancer tissues prompted us to detect whether it would be a tumor suppressor. Proliferation was measured by CCK8 kit after cells were transfected miR-100 mimics for 24, 48, 72 and 96h. No significant difference of proliferation of Hela(Fig. 4A) and Siha(Fig. 4B) were observed at the point of 24h after transfection. While at the other time points, proliferations were inhibited in cells treated with mimics.

Furthermore, the early apoptosis rates of Hela and Siha which transfected mimic and NC sequence were tested by annexin-V and PI assay and revealed together in Fig. 5A.

At last, cell cycle assay demonstrated that cell accounts in G2/M phase were decreased in Hela and Siha transfected miR-100 mimics respectively. Detail rates were shown in Fig. 5B.

MiR-100 inhibits tumorigenesis in SCC xenografts.

There mice were injected cells transfected with miR-100, while others injected without transfection. Compared to control group, the volumn of tumor was reduced (Fig. 6A&6B).

MiR-100 targets mTOR.

MiRanda, TargetScan, and miRbase predicted mTOR to be the target of miR-100.

According to sequence analysis, mTOR was found contains a putative binding site of miR-100 (Fig. 7A). To verify whether mTOR is the target of miR-100, a wild-type mTOR 3'UTR fragment was cloned downstream of the firefly luciferase reporter gene. When pc3-miR-100 was co-transfected, the relative luciferase activity of the reporter gene containing the wild-type 3'UTR was inhibited significantly Fig. 7B.And the expression of p-mTOR on cervical cancer was significantly higher than normal cervical Fig. 7C.

Mir-100 Was Provoked By 5-aza-cdr In Hela And Siha

We applied 10µM 5-Aza-CdR to HeLa and SiHa, to evaluate the effects on miR-100. As shown in Fig. 8A the treatment significantly up-regulated the level of miR-100 in both cell lines, suggesting a possible role of methylation in the regulation of miR-100 during the process of radiotherapy(P<0.05).

The level of miR-100 regulated by radiotherapy may not be mediated by miR-100HG

Bisulfite sequencing PCR was used to elevate the methylation of MIR100HG in HeLa, HRR, SiHa and SRR. The percentage of CpG Fig. 8B methylation was 0.207±0.050, 0.148±0.042,0.067±0.014, 0.045±0.021, respectively. As shown in Figure8B, the methylation of MIR100HG decreased after radiotherapy, though there was no statistic significance.

Circcasc15 May Sponge Mir100 In Cerna Manner

Three cervical cancer tissues and three normal cervical tissues were used for circRNAs expression profiling. The scatter and volcano plots demostrated the circRNA expression Fig. 9A. The cluster heat map showed circRNAs that expressed over 2 fold change. qPCR was performed to verify the microarray results. Consistent with the microarray, qPCR showed that circCASC15 was increased in Hela and Siha cells compared to normal cervical tissues (Fig. 9B).

With bioinformatic prediction, miR-100-5p was predicted as the sponge target of has-circ-CASC15 (Fig. 9C). To find the regulatory mechanism of miR-100, RNA pulldown assay was performed. CircCASC15 was found to be combined with miR-100(Fig. 9D). CircCasc15 and miR-100 were found mainly expressed in cytoplasm. Both in our microarray analysis and tissues’ validation, circCASC15 was downregulated (Fig. 9E).

In our study, the increase of miR-100 in the radio-resistant cell lines was detected, which was in accordance with the assay of breast cancer [18].While miR-100 was considered to be a suppressor gene, which used to decrease in cervical cancer. In this assay, its provoked expression suggested a potential role in sensitivity of radiotherapy. In oral cancer, the increase of miR-100 inhibited FGFBP1 and FGFR3, which over-expressed in radio-resistant cells [8], indirectly corroborate our hypothesis.

The accumulation of miR-100 altered in different fractionated radiation exposure patterns, namely longer interval and larger dose had better effect on inducing the expression, while the mechanism is still unknown.

It is recorded that DNA methylation may play an important role in silencing microRNA in tumors. A meta-analysis of 122 microRNAs showed that microRNAs regulated by methylation were mainly located in chromosome 1, 7, 11, 14 and 19[19]. As miR-100 locates in chromosome 11, we inferred that methylation may contribute to the regulation of it. Thereby we detected the effects of 5-Aza-CdR on HeLa and SiHa. With the treatment, the expressions of miR-100 were significantly induced. Our evidence linked methylation to irradiation on the increase of miR-100 in HeLa and SiHa, suggesting the level of methylation may have changed in the process of radiotherapy.

In breast cancer, miR-100 has been proved to be regulated by the methylation of MiR100HG, a host gene, where the miR-100 gene embedded[20]. Furthermore, we elevated the methylation of MiR100HG in SiHa, SR6, HeLa and HR6 to validate our hypothesis. The Bisulfite sequencing PCR showed that the percentage of CpG methylation slightly decreased in HR6 and SR6, compared to Hela and SiHa. Accordingly, the level of miR-100 regulated by radiotherapy may not be directly mediated by the methylation of MIR100HG. As the loss of chromosome 11 also can lead to the low expression of miR-100[21], the regulation of miR-100 remains to further research.

In conclusion, miR-100 turns out to be a tumor suppressor in cervical cancer for its exceeding ability to invasion /proliferation inhibition and apoptosis promoting. We’ve found that its inhibition of proliferation was double consequences, on one hand, miR-100 induced G2/M phase arrest which weakened cell mitosis, on the other hand, miR-100 initiated cell apoptosis which promoted cell death.

It’s widely shared that miRNAs could bind to 3’UTR leading mRNA cleavage or translation inhibition which negatively regulate downstream mRNA and transcription factors [22]. While as a single miRNA can regulate sorts of mRNAs and in return one individual mRNA could be controlled by several miRNAs. Nevertheless genes regulated by one miRNA often have similar functions. The identified targets of miR-100 include mTOR[7, 23] .They both act as cell cycle activative genes which enable and allow cells entry into mitosis[27–29].

MiR-100 influences cell cycle and apoptosis and it’s wildly demonstrated in ovary cancer[7], mammary carcinoma[20] and hepatocellular cancers[29] but its relationship to invasion is rarely reported. Our research found out miR-100 down-regulated invasiveness in cervical cancer cell lines for the first time. In the meanwhile, miR-100 could also reduce EMT, which explained its invasion cut down function. EMT is the characteristics that epithelial cells gaining mesenchyma transition, in this transforming cancer cells express MMPs including MMP2, MMP9, which is a family of key factors melting basement membrane and enable cells accessing circulation and mediate metastatic.

MTOR is a protein kinase belonging to PI3K family [30]. As the core component of many different complexes, mTOR plays a key role in a variety of biological processes [30–31]. Up-regulating mTOR signaling can promote tumor growth and progression through a variety of mechanisms [32]. One study has revealed increased expression of mTOR in cervical cancer [33]. In all cases, SCCs showed high translocation of p-mTOR and p-p70S6K in nuclear [34]. Faried et al. found activated AKT and mTOR related with poorer prognosis. The same authors [35] also investigated the expression of phosphorylated mTOR was independentand was a significant prognostic marker in cervical adenocarcinoma. In addition, the expression of p-mTORcan be used as a marker to predict chemotherapy response and survival of CC [36]. Moreover, Kim et al. [37] have also reported that p-mTOR was associated with poor response to radiotherapy. They [35] also independently studied the expression of p-mTOR as an important prognostic marker of cervical adenocarcinoma. In addition, the expression of phosphorylated mTOR can be used to predict the chemotherapy response and survival [36]. In addition, the cytoplasmic expression of p-mTOR was also related to the adverse reactions of radiotherapy [37].

CircCASC15 was also named hsa_circ_0075828. The circCASC15, generated from CASC15 gene, was 204 nucleotides in length. Divergent primers were designed and confirmed by Sanger sequencing. MRE (miRNA response element) has been proved existing in circRNA and acts as a competitive endogenous RNA (ceRNA) sponge miRNA or transcription regulator

In conclusion, circCASC15-miR-100-mTOR might influnce the EMT of cervical cancer. And this loop will be the target of therapy in the future.

Ethics approval and consent to participate

Use of patient tissue samples and nude mice were approved by the ethics committee of Sun Yat-sen Memorial Hospital

Consent to publish

Not applicable
Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.
Authors' contributions

Tingting Yaooriginally designed the project and wrote this article; Zhiliao Chenconceptualized the research; Yongpai Pengprepared the tables and figures; Guanglei ZhongFlorescent in situ hybridization. Chunxian Huangdid Transient transfection, Jing Li (F)did Tumor xenografts , Ruixin Li did 5-aza-2-deoxycytidine Treatment and BSP.All authors have read and approved the manuscript.

Acknowledgements

None.

Funding

This work was supported by National Natural Science Foundation of China (81572575),Guangdong province Natural Scientific Grant (2021A1515010267), Special supported for Guangdong College Students' innovation and entrepreneurship training program (1055813194),Guangdong clinical teaching base teaching program(2018JD004),CSCO(Y-2019AZMS-0393).

Baumann M, Krause M, Thames H, Trott K, Zips D. (2009). Cancer stem cells and radiotherapy. Int J Radiat Biol85:391-402.
Bergkvist GT, Argyle DJ, Pang LY, Muirhead R, Yool DA. (2011). Studies on the inhibition of feline EGFR in squamous cell carcinoma: enhancement of radiosensitivity and rescue of resistance to small molecule inhibitors. Cancer Biol Ther 11:927-937.
Beskow C, Skikuniene J, Holgersson A, Nilsson B, Lewensohn R, Kanter L, Viktorsson K. (2009). Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86. Br J Cancer 101:816-821.
Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT, Jiang J, Schmittgen TD, Patel T. Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology. 2006;130:2113–2129.
Yang H, Kong W, He L, Zhao JJ, O’Donnell JD, Wang J, Wenham RM, Coppola D, Kruk PA, Nicosia SV, Cheng JQ. MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. Cancer Res. 2008;68:425–433.
Xia L, Zhang D, Du R, Pan Y, Zhao L, Sun S, Hong L, Liu J, Fan D. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int J Cancer. 2008;123:372–379.
Nagaraja AK, Creighton CJ, Yu Z, et al. A link between mir-100 and FRAP1/mTOR in clear cell ovarian cancer. Mol Endocrinal,2010,24(2):447-463
Henson BJ, Bnattacharjee S, O’ Dee DM, et al. Decreased expression of miR-125b and miR-100 in oral cancer cells contributes to malignancy. Genes Chromosomes Cancer, 2009, 48(7):569-582.
Ng WL, Yan D, Zhang X, et al. Over-expression of miR-100 is responsible for the lower –expression of ATM in the human glioma cell line: M059J. DNA Repair(Amst), 2010,9(11):1170-1175.
Liu W, Gong YH, Chao TF, et al. Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas. Chin Med J(Engl), 2009, 122(20):2405-2411.
Wilusz JE, Sharp PA.Science. Molecular biology. A circuitous route to noncoding RNA. 2013 Apr 26; 340(6131):440-1.
Salzman J, Chen RE, Olsen MN, Wang PL, Brown PO.Cell-type specific features of circular RNA expression. PLoS Genet. 2013; 9(9):e1003777.
Circular RNA participates in the carcinogenesis and the malignant behavior of cancer.Zhao ZJ, Shen J.RNA Biol. 2017 May 4; 14(5):514-521.
Natural RNA circles function as efficient microRNA sponges.Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, Kjems J.Nature. 2013 Mar 21; 495(7441):384-8.
J. Koh, B.E. Greer, N.R. Abu-Rustum, S.M. Apte, S.M. Campos, J. Chan, K.R. Cho, D. Cohn, M.A. Crispens, N. DuPont, P.J. Eifel, D.K. Gaffney, R.L. Giuntoli 2nd, E. Han, W.K. Huh, J.R. Lurain 3rd, L. Martin, M.A. Morgan, Mutch, S.W. Remmenga, R.K. Reynolds, W. Small Jr., N. Teng, T. Tillmanns, F.A. Valea, N.R. McMillian, M. Hughes, National comprehensive cancer, cervicalcancer, J. Nat. Compr. Cancer Netw. e JNCCN 11 (2013) 320e343.
Huang C, Lu H, Li J, Xie X, Fan L, Wang D, Tan W, Wang Y, Lin Z, Yao T. SOX2 regulates radioresistance in cervical cancer via the hedgehog signaling pathway.Gynecol Oncol. 2018 Dec;151(3):533-541.
Fan L, Huang C, Li J, Gao T, Lin Z, Yao T.Long non coding RNA urothelial cancer associated 1 regulates radioresistance via the hexokinase 2/glycolytic pathway in cervical cancer.Int J Mol Med. 2018 Oct;42(4):2247-2259.
Adam Christopher Mueller, Dandan Sun, Anindya Dutta.The miR-99 family regulates the DNA damage response through its target SNF2.Oncogene,2013, 32 (9): 1164–1172.
Kunej T, Godnic I, Ferdin J, et al. Epigenetic regulation of microRNAs in cancer: an integrated review of literature.Mutat Res, 2011, 717(1-2):77-84.
Chen D, Sun Y, Yuan Y, et al. Mir-100 induces EMT but suppresses tumorgenesis, migration and invasion.PLoS Genet,2014, 10(2):e1004177.
SM Wilting, PJF Snijders, W Verlaat, et al.Altered microRNA expression associated with chromosomalchanges contributes to cervical carcinogenesis. Oncogene, 2013,32:106-116.
Chen CZ. MicroRNAs as oncogenes and tumor suppressors. N Engl J Med. 2005 Oct 27;353(17):1768-7.
Wang FZ, Weber F, Croce C, Liu CG, Liao X, Pellett PE. Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication. J Virol. 2008 Sep;82(18):9065-74.
Archambault V, Glover DM. Polo-like kinases: conservation and divergence in their functions and regulation. Nat Rev Mol Cell Biol. 2009 Apr;10(4):265-75.
Tarantino C, Paolella G, Cozzuto L, Minopoli G, Pastore L, Parisi S, Russo T.MiRNA 34a, 100, and 137 modulate differtiation of mouse embryonic stem cells. FASEB J. 2010 Sep;24(9):3255-63.
Rashmi R, DeSelm C, Helms C, Bowcock A, Rogers BE, Rader JL, Grigsby PW, Schwarz JK. AKT inhibitors promote cell death in cervical cancer through disruption of mTOR signaling and glucose uptake. PLoS One. 2014 Apr 4;9(4):e92948.
Boutros R, Lobjoois V, Ducommun B. CDC25 phosphatases in cancer cells:key players? Good targets? Nat Rev Cancer. 2007 Jul;7(7):495-507.
Strebhardt K. Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy. Nat Rev Drug Discov. 2010 Aug;9(8):643-60.
Petrelli A, Perra A, Schernhuber K et al. Sequential analysis of multistage hepatocarcinogenesis reveals that miR-100 and PLK1 dysregulation is an early event maintained along tumor progression. Oncogene 2012 10 18;31(42):4517-26.
Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–976.
Harwood FC, Klein Geltink RI, O’Hara BP, Cardone M, Janke L, Finkelstein D, et al. ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer. Sci Adv. 2018;4:eaar3938.
Yin Y, Hua H, Li M, Liu S, Kong Q, Shao T, et al. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR. Cell Res. 2016;26:46–65.
Ji J, Zheng PS.Activation of mTOR signaling pathway contributes to survival of cervical cancer cells. Gynecol Oncol. 2010 Apr; 117(1):103-8.
34.Feng W, Duan X, Liu J, Xiao J, Brown RE.Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.Int J Clin Exp Pathol. 2009; 2(3):249-60.
Kim M-N, Kim Y-J, Sung CO, Choi CH, Lee J-W, B-g Kim, et al. High expression of mTOR is associated with radiation resistance in cervical cancer. J Gynecol Oncol.2010;21:181–5.
de Melo AC, Paulino E, Garces ÁH.A Review of mTOR Pathway Inhibitors in Gynecologic Cancer. Oxid Med Cell Longev. 2017; 2017():4809751.
Tinker AV, Ellard S, Welch S, Moens F, Allo G, Tsao MS, et al. Phase II study of temsirolimus (CCI-779) in women with recurrent, unresectable, locally advanced or metastatic carcinoma of the cervix. A trial of the NCIC Clinical Trials Group (NCIC CTG IND 199). Gynecol Oncol 2013;130:269–74.

PDF

Reviews received at journal
05 Dec, 2021
Reviewers invited by journal
05 Dec, 2021
Editor invited by journal
30 Nov, 2021
Editor assigned by journal
29 Nov, 2021
First submitted to journal
22 Nov, 2021
Editorial decision: Major revision
10 Nov, 2021

You are reading this latest preprint version

CircCASC15-miR-100-mTOR may Influence the Cervical Cancer Radioresistance

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Patient selection and human tissues

Mirnas Real Time Polymerase Chain Reaction Array

Cell Culture

Transient Transfection

Mirna Expression Analysis And Rt-pcr Assay

Proliferation Assay

Cell Apoptosis Assay

Tumor Xenografts

Bioinformatics

Immunohistochemistry

Establishment Of Radioresistant (Rr) Cell Lines

5-aza-2-deoxycytidine Treatment

Bisulfite Sequencing Pcr(Bsp)

Results

Mir-100 Was Provoked By 5-aza-cdr In Hela And Siha

Circcasc15 May Sponge Mir100 In Cerna Manner

Discussion

Declarations

References

Status:

Version 1